Methods of modulating the negative chemotaxis of immune cells

ABSTRACT

The current invention is directed to methods of inducing migration of an immune cell toward a cancer cell comprising inhibiting the activity of a chemorepellant released from the cancer cell.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/572,445, filed Oct. 2, 2009, which claims the benefit of U.S. Provisional Application No. 61/102,177, filed Oct. 2, 2008 and U.S. Provisional Application No. 61/222,217 filed Jul. 1, 2009. The entire teachings of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

A long-standing dilemma in tumor immunology is the ability of solid tumor cells to escape immune surveillance despite demonstrable antitumor T-cell response. Primarily, the immune evasion mechanism of tumor has been evaluated in the context of expression of immunosuppressive bio-molecules viz., IL-10, transforming growth factor-b (TGF-b), indoleamine-2,3-deoxygenase (IDO), macrophage colony stimulating factor (M-CSF), arginase, prostaglandin E2 (PGE2), cyclooxygenase-2 (COX2) and nitric-oxide synthase 2 (NOS2), IL-6, chemokine CXCL12 and the like, that inhibit the function of dendritic cells (DC) and T cells. The increased expression of death inducing molecules (FasL & TRAIL), which induces apoptosis in tumor infiltrating T cells, has also been elucidated to explain the mechanism by which tumors evade the immune system.

The migration of immune cells to a target site is a major step in eliciting the immune response against tumor cell. Chemotaxis, or the oriented movement of a cell in response to a chemical agent, is a complex and highly integrated process. The movement can be positive (toward) or negative (away) from a chemical gradient. Movement toward an agent or stimulus is termed positive chemotaxis (i.e., the agent or stimulus is chemoattractive for the cell), while movement away from an agent or stimulus is termed negative chemotaxis (i.e., the agent or stimulus is chemorepulsive for the cell). It is believed that for both prokaryotes and eukaryotes, cells undergoing chemotaxis sense a change in agent concentration and, thereby, move in response to the concentration gradient. Chemoattraction (CA) and chemorepulsion (CR) are therefore properties of the agent or stimulus, while chemotaxis is a property of cells.

The present inventors have discovered proteins which are expressed (secreted) by tumor cells which keep anti-tumor T cells (CD4 & CD8), neutrophils, NK cells at the bay while concomitantly recruiting regulatory T cells at tumor sites and thus mediating evasion of the immune response. It would be advantageous to identify these proteins released from cancer cells that induce negative chemotaxis of immune cells and/or inhibit the activity of these proteins in order to induce positive chemotaxis of immune cells toward cancer cells.

SUMMARY OF THE INVENTION

The present invention provides methods of inducing the migration of an immune cell toward a cancer cell comprising inhibiting the activity of a chemorepellant released from the cancer cell.

In some embodiments, the activity of a chemorepellant released from a human cancer cell is inhibited. In other embodiments, the human cancer cell is selected from the group consisting of a renal adenocarcinoma cell, renal carcinoma cell, a glioblastoma cell a colon carcinoma cell, a hepatocellular carcinoma cell, an ovarian carcinoma cell and a prostate cancer cell.

In one embodiment, the activity of a chemorepellant released from the cancer cell is inhibited, wherein the chemorepellant comprises a sequence that has substantial identity to a protein isolated from ovarian cancer cystic fluid or to a biologically active fragment thereof, wherein the isolated protein or fragment thereof is capable of inducing chemorepulsion of an immune cell. In another embodiment, the chemorepellant comprises a sequence that has substantial identity to a protein isolated from a supernatant of a cell line or to a biologically active fragment thereof, wherein the cell line is selected from the group consisting of a human renal adenocarcinoma cell, a human renal carcinoma cell, a human glioblastoma cell, a human colon carcinoma cell, a human hepatocellular carcinoma cell, a human ovarian carcinoma cell and a human prostate cancer cell.

In another embodiment, the chemorepellant has substantial identity to the protein isolated from an ovarian cystic fluid, or to a biologically active fragment thereof. In another embodiment, the chemorepellant has substantial identity to of a protein isolated from a supernatant of a cell line, or a biologically active fragment thereof, wherein the cell line is selected from the group consisting of a human renal adenocarcinoma cell, a human renal carcinoma cell, a human glioblastoma cell, a human colon carcinoma cell, a human hepatocellular carcinoma cell, a human ovarian carcinoma cell and a human prostate cancer cell.

In one embodiment, the chemorepellant has substantial identity to a protein selected from a chemorepellant protein set forth in Tables 1 to 9, or a biologically active fragment of thereof. In an additional embodiment, the chemorepellant has substantial identity to a protein selected from a protein set forth in Table 10 to 11, or a biologically active fragment thereof. In another embodiment, the chemorepellant has substantial identity to a protein selected from the group selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein, or a biologically active fragment of any of thereof.

In yet another embodiment, the invention is a method of treating cancer in a patient in need thereof comprising inhibiting the activity of a chemorepellant released from a cancer cell.

In a further embodiment, the invention is a method of inducing negative chemotaxis of a human immune cell comprising administering an inventive chemorepellant. In some embodiments, the chemorepellant comprises a sequence that has substantial identity to a protein isolated from ovarian cancer cystic fluid or to a biologically active fragment thereof, wherein the isolated protein or fragment thereof is capable of inducing chemorepulsion of an immune cell. In another embodiment, the invention is a method of inducing negative chemotaxis of a human immune cell comprising administering a chemorepellant, wherein the chemorepellant comprises a sequence that has substantial identity to a protein isolated from a supernatant of a cell line selected from the group consisting of a human renal adenocarcinoma cell, a human renal carcinoma cell, a human glioblastoma cell, a human colon carcinoma cell, a human hepatocellular carcinoma cell, a human ovarian carcinoma cell and a human prostate cancer cell, or a biologically active fragment of said isolated protein, wherein said protein or fragment thereof is capable of inducing negative chemotaxis. In an additional embodiment, the administered chemorepellant comprises a sequence that has substantial identity to a protein listed in Tables 1 to 9, or to a biologically active fragment thereof. In an additional embodiment, the administered chemorepellant has substantial identity to a protein listed in Tables 10 to 11, or to a biologically active fragment thereof. In yet another embodiment, the administered chemorepellant comprises a sequence that has substantial identity to a protein selected from the group selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein, or a biologically active fragment thereof.

In yet another embodiment, the invention is a method of treating a condition mediated by migration of a human migratory cell toward a chemotactic site comprising administering to said patient a therapeutically effective amount of an inventive chemorepellant. In some embodiments, the chemorepellant comprises a sequence that has substantial identity to a protein isolated from ovarian cancer cystic fluid, or to a biologically active fragment thereof, wherein the isolated protein or fragment thereof is capable of inducing chemorepulsion of an immune cell. In a further embodiment, the invention is a method of treating a condition mediated by migration of a human migratory cell toward a chemotactic site comprising administering to said patient a therapeutically effective amount of a chemorepellant, wherein said chemorepellant comprises a sequence that has substantial identity to a protein isolated from a supernatant of a cell line selected from the group consisting of a human renal adenocarcinoma cell, a human renal carcinoma cell, a human glioblastoma cell, a human colon carcinoma cell, a human hepatocellular carcinoma cell, a human ovarian carcinoma cell and a human prostate cancer cell, or to a biologically active fragment of any of thereof, wherein the protein or fragment thereof is capable of inducing negative chemotaxis of an immune cell.

These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the drawings and the detailed description of the embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIG. 1 is a bar graph showing fold induction (over media) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 1:30, 1:10, 1:3 and neat dilutions of ovarian cancer cyst fluid.

FIG. 2 is a bar graph showing fold induction (over media) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with S-200 chromatography fractions of cystic fluids.

FIG. 3 is a bar graph showing fold induction (over media) of chemorepulsion of neutrophils treated with 0.0011, 0.011, 0.11 and 1.1 uM actin (left) and 0.0018, 0.018, 0.18 and 1.8 uM 14-3-3 (right).

FIG. 4 is a bar graph showing fold induction (over media) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with a 1:1 combination of actin and 14-3-3 at 1:27, 1:9, 1:3 and neat dilutions.

FIG. 5 is a bar graph showing fold induction (over media) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.0051, 0.051, 0.51 and 5.1 uM apolipoprotein A1.

FIG. 6 is a bar graph showing fold induction (over media) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.0088, 0.088, 0.88 and 8.8 uM hemopexin.

FIG. 7 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.15, 0.46, 1.39 and 4.16 uM Park-7.

FIG. 8 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.28, 0.77, 2.30 and 6.9 uM cofilin-1.

FIG. 9 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.43, 1.28, 3.83 and 11.48 uM 14-3-3 epsilon.

FIG. 10 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.13, 0.39, 1.18, 3.53 uM 14-3-3 gamma.

FIG. 11 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.15, 0.44, 1.33 and 3.99 uM phosphoserine phosphatase.

FIG. 12 is a bar graph showing fold induction (over media) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.32, 0.97, 2.92 and 8.76 uM superoxide dismutase.

FIG. 13 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.28, 0.85, 2.56 and 7.68 uM profilin-2.

FIG. 14 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.45, 1.36, 4.07 and 12.20 uM beta-2 microglobulin.

FIG. 15 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 3, 9, 27 and 81.1 uM cytochrome C.

FIG. 16 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.22, 0.66, 1.98 and 5.95 uM cystatin B.

FIG. 17 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.16, 0.48, 1.43 and 4.3 uM macrophage inhibitor factor (MIF).

FIG. 18 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.47, 1.41, 4.23 and 12.70 uM FKBP.

FIG. 19 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 0.16, 0.48, 1.43 and 12.2 uM thioredoxin.

FIG. 20 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with ACHN supernatant fractions collected from day 0 (d0) to day 4 (d4) and Turbodoma (used as controls).

FIGS. 21A and B are bar graphs showing induction (number of cells per well) of chemorepulsion (B) and chemoattraction (A) of neutrophils treated with ACHN size exclusion fractions.

FIG. 22 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with 786-O supernatant fractions collected from day 0 (d0) to day 4 (d4) and Turbodoma control (TD).

FIGS. 23A and B are bar graphs showing induction (number of cells per well) of chemorepulsion (B) and chemoattraction (A) of neutrophils treated with 786-O size exclusion fractions.

FIG. 24 is a photograph of the SDS PAGE gel of supernatant fractions from ACHN and 786-O.

FIG. 25 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with SF-359 supernatant fractions collected from day 2 (d2) to day 4 (d4) and TD control.

FIGS. 26A and B are bar graphs showing fold induction (over media) (A) or number of cells (B) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with SF-359 size exclusion fractions.

FIG. 27 is a photograph of the SDS PAGE gel of supernatant fractions from SF-359 culture.

FIG. 28 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) of neutrophils treated with U-251 supernatant fractions collected from day 0 (d0) to day 4 (d4) and TD control.

FIGS. 29A and B are bar graphs showing induction (number of cells per well) of chemoattraction (A) and chemorepulsion (B) of neutrophils treated with U-251 size exclusion fractions.

FIG. 30 is a photograph of an SDS PAGE gel of supernatant fractions from U-251 supernatant fractions.

FIG. 31 is a bar graph showing induction (number of cells per well) of chemorepulsion (right) and chemoattraction (left) treated with HCC-2998 supernatants collected from day 0 (d0) to day 4 (d4) and TD control.

FIGS. 32A and 32B are bar graphs showing induction (number of cells per well) of chemoattraction (A) and chemorepulsion (B) of neutrophils treated with HCC-2998 size exclusion fractions.

FIG. 33 is a bar graph showing fold induction (over media) of chemoattraction (left) and chemorepulsion (right) of HepG2 supernatant fractions collected from day 0 (d0) to day 7 (d7).

FIG. 34 is a bar graph showing fold induction (over media) of chemoattraction (left) and chemorepulsion (right) of HepG2 size exclusion fractions.

FIG. 35 is a bar graph showing fold induction (over media) of chemoattraction (left) or chemorepulsion (right) of neutrophils treated with CRL-1978 supernatants collected from day 0 (d0) to day 7 (d7) and TD control.

FIG. 36 is a bar graph showing fold induction (over media) of chemoattraction (left) or chemorepulsion (right) of neutrophils treated with CRL-1978 size exclusion fractions.

FIG. 37 is a bar graph showing fold induction (over media) of chemoattraction (left) or chemorepulsion (right) of neutrophils treated with PC3 supernatants from day 0 (d0) to day 7 (d7) and TD control.

FIG. 38 is a bar graph showing fold induction (over media) of chemoattraction (left) or chemorepulsion (right) of neutrophils treated with PC3 size exclusion fractions.

FIG. 39 is a bar graph showing RU of chemoattraction (left) and chemorepulsion (right) of neutrophils treated with SK-BR-3 anion exchange fractions (A2-A8) and media.

FIG. 40 is a photograph of a gel (Comassie Stain) of SK-BR-3 anion exchange fractions submitted for mass spectrometry (MS) analysis.

DETAILED DESCRIPTION OF THE INVENTION

A description of the embodiments of the invention follows.

As used herein, “a” or “an” are taken to mean one or more unless otherwise specified.

The present invention is based on the surprising discovery that one or more proteins isolated from ovarian cancer cystic fluid and/or from the supernatants of human cancer cell cultures induce negative chemotaxis of neutrophils. For example, as shown in Example 1, neutrophils contacted with certain chromatographic fractions of ovarian cancer cystic fluid showed greater than 9-fold induction of chemotaxis than that in response to media.

In one embodiment, the invention is a method of inducing migration of an immune cell toward a cancer cell comprising inhibiting the activity of a chemorepellant released from the cancer cell. In some embodiments, the cancer cell is selected from the group consisting of colon carcinoma cell, prostate cancer cell, breast cancer cell, lung cancer cell, skin cancer cell, liver cancer cell, bone cancer cell, pancreas cancer cell, ovarian cancer cell, testicular cancer cell, bladder cancer cell, kidney cancer cell, brain cancer cell, glioma cell, head and neck cancer cell. In another embodiment, the cancer cell is a renal adenocarcinoma cell, renal carcinoma cell, a glioblastoma cell a colon carcinoma cell, a hepatocellular carcinoma cell, an ovarian carcinoma cell and a prostate cancer cell.

According to the present method, migration of an immune cell toward a cancer cell can be induced by inhibiting the activity of a chemorepellant released from the cancer cell. The chemorepellant released from the cancer cell is a protein that induces negative chemotaxis of an immune cell. The inventive methods also encompass a method of inducing negative chemotaxis of an immune cell comprising administering a chemorepellant, wherein the chemorepellant comprises a sequence that has substantial identity to a protein release from a cancer cell, or a biologically active fragment thereof.

A “chemorepellant” is an agent or stimulus that induces, elicits or triggers negative chemotaxis of a migratory cell (movement away from an agent or stimulus). In one embodiment, the chemorepellant comprises an amino acid sequence that has substantial identity to a protein isolated from ovarian cancer cystic fluid, or to a biologically active fragment thereof, wherein the isolated protein or fragment thereof is capable of inducing chemorepulsion of an immune cell. In another embodiment, the chemorepellant has substantial identity to a protein isolated from ovarian cancer cystic fluid or to a biologically active fragment thereof. In an additional embodiment, the chemorepellant has substantial identity to a protein isolated from ovarian cancer cystic fluid.

In another embodiment, the chemorepellant comprises a sequence that has substantial identity to a protein isolated from a supernatant of a cell line selected from the group consisting of a human renal adenocarcinoma cell, a human renal carcinoma cell, a human glioblastoma cell, a human colon carcinoma cell, a human hepatocellular carcinoma cell, a human ovarian carcinoma cell and a human prostate cancer cell, or a biologically active fragment of said isolated protein, wherein said protein or fragment thereof is capable of inducing negative chemotaxis. In yet another embodiment, the chemorepellant has substantial identity to a protein isolated from a supernatant of a cell line selected from the group consisting of a human renal adenocarcinoma cell, a human renal carcinoma cell, a human glioblastoma cell, a human colon carcinoma cell, a human hepatocellular carcinoma cell, a human ovarian carcinoma cell and a human prostate cancer cell, or a biologically active fragment of said isolated protein.

In yet another embodiment, the chemorepellant comprises a sequence that has substantial identity to a protein set forth in Tables 1 through 9 (shown below in Examples 1 to 3), or to a biologically active fragment thereof. In a further embodiment, the chemorepellant has substantial identity to a protein set forth in Tables 1 through 9, or a biologically active fragment thereof. In yet another embodiment, the chemorepellant is a protein set forth in Tables 1 through 9. In another embodiment, the chemorepellant is a protein set forth in Tables 10 to 11.

In an additional embodiment, the chemorepellant protein is a protein that is released by at least two distinct cancer cells. Cancer cells are distinct when they are of different origin or different cancer cell types. For example, liver cancer cells and ovarian cancer cells are distinct cancer cells. Similarly, a cancer cell of the kidney cancer cell line, ACHN, is distinct from the kidney cancer cell line 786-O. In a further embodiment, the chemorepellant protein has substantial identity to a protein set forth in Tables 10-11, or to a biologically active fragment thereof.

In another embodiment, the chemorepellant comprises a sequence that has substantial identity to the amino acid sequence of a protein selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein, or a biologically active fragment of any of thereof. In an additional embodiment, the chemorepellant has substantial identity to a protein selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein. In a further embodiment, the chemorepellant is a protein selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein. Accession Numbers for these proteins are shown below in Tables 1 through 9.

A biologically active fragment is a peptide fragment of a naturally occurring protein or the full-length protein that retains at least some of the biological activity of the naturally occurring protein or the full-length protein. In some embodiments, the biological activity is the ability to induce chemorepulsion of a human migratory cell.

Ovarian cancer cystic fluid refers to cystic fluid from patients with ovarian carcinomas.

In some embodiments, the chemorepellant comprises a sequence that has substantial identity to a protein isolated from the supernatant of a cancer cell culture, wherein the culture is of a human cancer cell selected from the group consisting of a renal adenocarcinoma cell, renal carcinoma cell, a glioblastoma cell a colon carcinoma cell, a hepatocellular carcinoma cell, an ovarian carcinoma cell and a prostate cancer cell. In one embodiment, the human renal adenocarcinoma cell line is ACHN. In another embodiment, the human renal carcinoma cell line is 786-O. In another embodiment, the human glioblastoma cell line is SF539 or U251. In an additional embodiment, the human colon carcinoma cell line is HCC-2998. In a further embodiment, the human hepatocellular carcinoma cell line is HepG2 (ATCC No. HB-8065). In yet another embodiment, the human ovary clear cell carcinoma cell line is ATCC No. CRL-1978. In an additional embodiment, the human prostate cancer cell line is PC3 (ATCC No. CRL-1435).

In certain embodiments of the invention, the chemorepellant comprises a sequence that has substantial identity to the amino acid sequence of a protein isolated from ovarian cancer cystic fluid or the supernatant of a cancer cell line. In these embodiments, the ovarian cancer cystic fluid or supernatant is fractionated and the protein is isolated from a chemorepulsive fraction. A chemorepulsive fraction is a fraction that induces chemorepulsion of a human migratory cell. The ovarian cystic fluid or supernatant can be fractionated, for example, by size exclusion and anion exchange chromatography.

Exemplary amino acid sequences for actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein are shown below:

Actin (IPI Acc. No. IPI100021439 (+2)) (SEQ ID NO: 1) MDDDIAALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVG DEAQSKRGILTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPK ANREKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVTHTVPIYEGYAL PHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMATA ASSSSLEKSYELPDGQVITIGNERFRCPEALFQPSFLGMESCGIHETTFNSIMKCDVDIRK DLYANTVLSGGTTMYPGIADRMQKEITAL APSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKQEYDESGPSIVHRKCF 14-3-3 (IPI Acc. No. IPI100021263 (+1)) (SEQ ID NO: 2) MDKNELVQKAKLAEQAERYDDMAACMKSVTEQGAELSNEERNLLSVAYKNVVGAR RSSWRVVSSIEQKTEGAEKKQQMAREYREKIETELRDICNDVLSLLEKFLIPNASQAES KVFYLKMKGDYYRYLAEVAAGDDKKGIVDQSQQAYQEAFEISKKEMQPTHPIRLGLA LNFSVFYYEILNSPEKACSLAKTAFDEAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSD TQGDEAEAGEGGEN GLLPVLESFK VSFLSALEEY TKKLNTQ Apoliprotein A1 (SwissProt Acc. No. P02647) (SEQ ID NO: 3) MKAAVLTLAV LFLTGSQARH FWQQDEPPQSPWDRVKDLATVYVDVLKDSG RDYVSQFEGSALGKQLNLKL LDNWDSVTST FSKLREQLGP VTQEFWDNLE KETEGLRQEM SKDLEEVKAKVQPYLDDFQK KWQEEMELYR QKVEPLRAEL QEGARQKLHE LQEKLSPLGE EMRDRARAHVDALRTHLAPYSDELRQRLA ARLEALKENGG ARLAEYHAKA TEHLSTLSEK AKPALEDLRQ Hemopexin (SwissProt Acc. No. P02790) (SEQ ID NO: 4) MARVLGAPVA LGLWSLCWSL AIATPLPPTS AHGNVAEGET KPDPDVTERC SDGWSFDATTLDDNGTMLFF KGEFVWKSHK WDRELISERW KNFPSPVDAA FRQGHNSVFL IKGDKVWVYPPEKKEKGYP LLQDEFPGIP SPLDAAVECH RGECQAEGVL FFQGDREWFW DLATGTMKERSWPAVGNCSS ALRWLGRYYC FQGNQFLRFD PVRGEVPPRY PRDVRDYFMP CPGRGHGHRN GTGHGNSTHH GPEYMRCSPH LVLSALTSDNHGATYAFSGT HYWRLDTSRD GWHSWPIAHQWPQGPSAVDA AFSWEEKLYL VQGTQVYVFL TKGGYTLVSG YPKRLEKEVG TPHGIILDSVDAAFICPGSS RLHIMAGRRL WWLDLKSGAQ ATWTELPWPH EKVDGALCME KSLGPNSCSANGPGLYLIHG PNLYCYSDVE KLNAAKALPQ PQNVTSLLGC TH PARK-7 DJ1 (IPI Acc. No. IPI00298547) (SEQ ID NO: 5) MASKRALVILAKGAEEMET IPVDVMRRAG IKVTVAGLAGKDPVQCSRDV VICPDASLED AKKEGPYDVVVLPGGNLGAQNLSESAAVKEILKEQENRKG LIAAICAGPTALLAHEIGFGSKVTTHPLAKDKMMNGGHYTYSENRVEKDG LILTSRGPGTSFEFALAIVEALNGKEVAAQVKAPLVLKD Cofilin-1 (IPI Acc. No IPI00012011) (SEQ ID NO: 6) MASGVAVSDG VIKVFNDMKV RKSSTPEEVK KRKKAVLFCL SEDKKNIILE EGKEILVGDV GQTVDDPYAT FVKMLPDKDC RYALYDATYE TKESKKEDLV FIFWAPESAP LKSKMIYASS KDAIKKKLTG IKHELQANCY EEVKDRCTLA EKLGGSAVIS LEGKPL 14-3-3 epsilon (IPI Acc. No. IPI00000816) (SEQ ID NO: 7) MDDREDLVYQ AKLAEQAERY DEMVESMKKV AGMDVELTVE ERNLLSVAYK NVIGARRASW RIISSIEQKEENKGGEDKLK MIREY RQMVE TELKLICCDI LDVLDKHLIP AANTGESKVF YYKMKGDYHR YLAEFATGND RKEAAENSLV AYKAASDIAM TELPPTHPIR LGLALNFSVF YYEILNSPDR ACRLAKAAFD DAIAELDTLS EESYKDSTLI MQLLRDNLTL WTSDMQGDGE EQNKEALQDV EDENQ 14-3-3- gamma (SwissProt. Acc. No. P61981; IPI Acc. No.IPI00220642) (SEQ ID NO: 8) MVDREQLVQKARLAEQAERYDDMAAAMKNVTELNEPLSNEERNLLSVAYKNVVGA RRSSWRVISSIEQKTSADGNEKKIEMVRAYREKIEKELEAVCQDVLSLLDNYLIKNCSE TQYESKVFYLKMKGDYYRYLAEVATGEKRATVVESSEKAYSEAHEISKEHMQPTHPIR LGLALNYSVFYYEIQNAPEQACHLAKTAFDDAIAELDTLNEDSYKDSTLIMQLLRDNL TLWTSDQQDDDGGEGNN Phosphoserine Phosphatase, (IPI Acc. No. IPI00019178; UNIPROT Acc. No. Q5EY1) (SEQ ID NO: 9) MVSHSELRKL FYSADAVCFD VDSTVIREEG IDELAKICGV EDAVSEMTRR AMGGAVPFKA ALTERLALIQ PSREQVQRLI AEQPPHLTPG IRELVSRLQE RNVQVFLISG GFRSIVEHVA SKLNIPATNV FANRLKSYFN GEYAGFDETQ PTAESGGKGE VIKLLKEKFH FKKIIMIGDG ATDMEACPPA DAFIGFGGNV IRQQVKDNAK WYITDFVELL GELEE Superoxide dismutase (IPI Acc. No. IPI00218733) (SEQ ID NO: 10) MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAG CTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIED SVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ Profilin-2 (IPI Acc. No. IPI00219468) (SEQ ID NO: 11) MAGWQSYVDNLMCDGCCQEAAIVGYCDAKYVWAATAGGVFQSITPIEIDMIVGKDR EGFFTNGLTLGAKKCSVIRDSLYVDGDCTMDIRTKSQGGEPTYNVAVGRAGRVLVFV MGKEGVHGGGLNKKAYSMAKYLRDSGF Beta-2 microglobulin (IPI Acc. No. IPI00004656) (SEQ ID NO: 12) MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDL LKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDR DM Cytochrome C (IPI Acc. No. IPI100465315) (SEQ ID NO: 13) MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNK GIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE Cystatin B (IPI Acc. No. IPI00021828) (SEQ ID NO: 14) MMCGAPSATQ PATAETQHIA DQVRSQLEEK ENKKFPVFKA VSFKSQVVAG TNYFIKVHVGDEDFVHLRVF QSLPHENKPL TLSNYQTNKA KHDELTYF Macrophage migration inhibitory factor (MIF) (IPI Acc. No. IPI00293276) (SEQ ID NO: 15) MPMFIVNTNVPRASVPDGFLSELTQQLAQATGKPPQYIAVHVVPDQLMAFGGSSEPCA LCSLHSIGKIGGAQNRSYSKLLCGLLAERLRISPDRVYINYYDMNAANVGWNNSTFA FK506 binding protein (IPI Acc. No IPI00873810) (SEQ ID NO: 16) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE Thioredoxin (IPI Acc. No. IPI00216298) (SEQ ID NO: 17) MVKQIESKTA FQEALDAAGD KLVVVDFSAT WCGPCKMIKP FFHSLSEKYS NVIFLEVDVD DCQDVASECE VKCMPTFQFF KKGQKVGEFS GANKEKLEAT INELV Galectin 3 (IPI Acc. No. IPI00465431) (SEQ ID NO: 18) MADNFSLHDA LSGSGNPNPQ GWPGAWGNQP AGAGGYPGAS YPGAYPGQAP PGAYPGQAPP GAYPGAPGAY PGAPAPGVYP GPPSGPGAYP SSGQPSATGA YPATGPYGAP AGPLIVPYNL PLPGGVVPRM LITILGTVKP NANRIALDFQ RGNDVAFHFN PRFNENNRRV IVCNTKLDNN WGREERQSVF PFESGKPFKI QVLVEPDHFK VAVNDAHLLQ YNHRVKKLNE ISKLGISGDI DLTSASYTMI Transferrin (TRFE_HU Serotransferrin precursor) (Acc. No. P02787) (SEQ ID NO: 19) MRLAVGALLV CAVLGLCLAV PDKTVRWCAV SEHEATKCQS FRDHMKSVIP SDGPSVACVK KASYLDCIRA IAANEADAVT LDAGLVYDAY LAPNNLKPVV AEFYGSKEDP QTFYYAVAVV KKDSGFQMNQ LRGKKSCHTG LGRSAGWNIP IGLLYCDLPE PRKPLEKAVA NFFSGSCAPC ADGTDFPQLC QLCPGCGCST LNQYFGYSGA FKCLKDGAGD VAFVKHSTIF ENLANKADRD QYELLCLDNT RKPVDEYKDC HLAQVPSHTV VARSMGGKED LIWELLNQAQ EHFGKDKSKE FQLFSSPHGK DLLFKDSAHG FLKVPPRMDA KMYLGYEYVT AIRNLREGTC PEAPTDECKPVKWCALSHHE RLKCDEWSVN SVGKIECVSA ETTEDCIAKI MNGEADAMSL DGGFVYIAGK CGLVPVLAEN YNKSDNCEDT PEAGYFAVAV VKKSASDLTW DNLKGKKSCH TAVGRTAGWN IPMGLLYNKI NHCRFDEFFS EGCAPGSKKD SSLCKLCMGS GLNLCEPNNK EGYYGYTGAF RCLVEKGDVA FVKHQTVPQN TGGKNPDPWA KNLNEKDYEL LCLDGTRKPV EEYANCHLAR APNHAVVTRK DKEACVHKIL RQQQHLFGSN VTDCSGNFCL FRSETKDLLF RDDTVCLAKLHDRNTYEKYL GEEYVKAVGN LRKCSTSSLL EACTFRRP EF-1-gamma (EF1G-HU Elongation factor 1-gamma) (Acc. No. P26641) (SEQ ID NO: 20) MAAGTLYTYP ENWRAFKALI AAQYSGAQVR VLSAPPHFHF GQTNRTPEFL RKFPAGKVPA FEGDDGFCVF ESNAIAYYVS NEELRGSTPE AAAQVVQWVS FADSDIVPPA STWVFPTLGI MHHNKQATEN AKEEVRRILG LLDAYLKTRT FLVGERVTLA DITVVCTLLW LYKQVLEPSF RQAFPNTNRW FLTCINQPQF RAVLGEVKLC EKMAQFDAKK FAETQPKKDT PRKEKGSREE KQKPQAERKE EKKAAAPAPE EEMDECEQAL AAEPKAKDPF AHLPKSTFVL DEFKRKYSNE DTLSVALPYF WEHFDKDGWS LWYSEYRFPE ELTQTFMSCN LITGMFQRLD KLRKNAFASV ILFGTNNSSS ISGVWVFRGQ ELAFPLSPDW QVDYESYTWR KLDPGSEETQ TLVREYFSWE GAFQHVGKAF NQGKIFK Galectin-3 binding protein (LG3BP_HU galectin 3 binding protein precursor) (Acc. No. Q08380 (SEQ ID NO: 21) MTPPRLFWVW LLVAGTQGVN DGDMRLADGG ATNQGRVEIF YRGQWGTVCD NLWDLTDASV VCRALGFENA TQALGRAAFG QGSGPIMLDE VQCTGTEASL ADCKSLGWLK SNCRHERDAG VVCTNETRST HTLDLSRELS EALGQIFDSQ RGCDLSISVN VQGEDALGFC GHTVILTANL EAQALWKEPG SNVTMSVDAE CVPMVRDLLR YFYSRRIDIT LSSVKCFHKL ASAYGARQLQ GYCASLFAIL LPQDPSFQMP LDLYAYAVAT GDALLEKLCL QFLAWNFEAL TQAEAWPSVP TDLLQLLLPR SDLAVPSELA LLKAVDTWSW GERASHEEVE GLVEKIRFPM MLPEELFELQ FNLSLYWSHE ALFQKKTLQA LEFHTVPFQL LARYKGLNLT EDTYKPRIYT SPTWSAFVTD SSWSARKSQL VYQSRRGPLV KYSSDYFQAP SDYRYYPYQS FQTPQHPSFL FQDKRVSWSL VYLPTIQSCW NYGFSCSSDE LPVLGLTKSG GSDRTIAYEN KALMLCEGLF VADVTDFEGW KAAIPSALDT NSSKSTSSFP CPAGHFNGFR TVIRPFYLTN SSGVD

As used herein, a chemorepellant has “substantial identity” to another protein when the chemorepellant has an amino acid sequence that has at least about 60 percent sequence identity, at least about 70 percent sequence identity, at least about 80 percent sequence identity, at least about 85 percent sequence identity, at least about 85 to 95 percent sequence identity, at least about 90 to about 95 percent sequence identity, at least about 98 percent sequence identity, or at least about 99 percent sequence identity to the amino acid sequence of the other protein. The terms “sequence identity” or “identity” in reference to a sequence refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. The terms “sequence homology” or “homology” in reference to a sequence refers to sequence homology between two amino acid sequences or two nucleotide sequences. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.

A “chemoattractant” is an agent or stimulus that induces, elicits or triggers positive chemotaxis (movement towards an agent or stimulus) by a migratory cell.

As used herein the terms “induce,” “elicit,” and “trigger,” when referring to the activity of a chemorepellant or chemoattractant with respect to negative or positive chemotaxis, carry the same meaning.

The activity of the chemorepellant released from a cancer cell is inhibited when the ability of the chemorepellant to induce negative chemotaxis of the immune cell is suppressed or decreased. According to the current invention, the activity of the chemorepellant released from the cancer cell can be inhibited by any means that suppresses negative chemotaxis of the immune cell or that induces positive chemotaxis of the immune cell toward the cancer cell. For example, the activity of the chemorepellant can be inhibited by administering an agent that inhibits the activity of the chemorepellants. Such agents, include, but are not limited to, small molecules, proteins, antibodies, and antisense nucleic acids.

In one embodiment, the activity of the chemorepellant released from a cancer cell is inhibited when the release of the chemorepellant is suppressed or decreased.

In another embodiment, the activity of the chemorepellant released from a cancer cell is inhibited by administering an agent that binds to the chemorepellant and inhibits its activity. In some embodiments, the activity of the chemorepellant is inhibited by administering an antibody that binds the chemorepellant and inhibits chemorepellant activity. The term “antibody” as used herein refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The term antibody, as used herein, includes antibody fragments either produced by modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) (scFv) or those identified using phase display libraries (see, for example, McCafferty et al. (1990) Nature 348:552-554). The term antibody also encompasses both monoclonal and polyclonal antibodies. The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production. In one embodiment, the antibody does not bind other proteins or molecules other than the chemorepellant.

Antibodies can be raised against an appropriate immunogen, including a chemorepellant released from a cancer cell or a fragment thereof. Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique. A variety of methods have been described (see e.g., Kohler et al., Nature, 256:495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976); Milstein et al., Nature 266:550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.); and Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, 1991); the teachings of each of which are incorporated herein by reference). Other suitable methods of producing or isolating antibodies of the requisite specificity can used, including, for example, methods which select recombinant antibody from a library, or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies (see e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 2555 (1993); Jakobovits et al., Nature, 362:255 258 (1993); Lonberg et al., U.S. Pat. No. 5,545,806; and Surani et al., U.S. Pat. No. 5,545,807; the teachings of which are each incorporated herein by reference). Single-chain antibodies, and chimeric, humanized or primatized (CDR-grafted), or veneered antibodies, as well as chimeric, CDR-grafted or veneered single-chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term “antibody.” The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0 125 023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0 120 694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0 194 276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0 239 400 B1; Queen et al., European Patent No. 0 451 216 B1; and Padlan et al., EP 0 519 596 A1. See also, Newman et al., BioTechnology, 10:1455 1460 (1992), regarding primatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 and Bird et al., Science, 242:423 426 (1988) regarding single-chain antibodies. In addition, antigen-binding fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single-chain antibodies, can also be produced, including, but not limited to, Fv, Fab, Fab′ and F(ab′)₂ fragments are encompassed by the invention.

In another embodiment, the activity of the chemorepellant is inhibited by administering an antisense nucleic acid. In this context, the chemorepellant antisense nucleic acid comprises at least six nucleotides that are antisense to a gene or cDNA encoding the chemorepellant released from a cancer cell or a portion thereof. The antisense nucleic acid is capable of hybridizing to a portion of an RNA encoding the chemorepellant. The antisense nucleic acid is a double-stranded or single-stranded oligonucleotide, RNA or DNA or a modification or derivative thereof, and can be directly administered to a cell or produced intracellularly by transcription of exogenous, introduced sequences. In one embodiment, the antisense nucleic acid has from about 6 to about 50 nucleotides. In other embodiment, the antisense nucleic acid has at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides. The antisense nucleic acid can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof and can be single-stranded or double-stranded. In addition, the antisense molecules can be polymers that are nucleic acid mimics, such as PNA, morpholino oligos, and LNA. Other types of antisense molecules include short double-stranded RNAs, known as siRNAs, and short hairpin RNAs, and long dsRNA (greater than 50 base pairs).

In yet another embodiment, the activity of the chemorepellant is inhibited by administering a ribozyme molecule that is designed to catalytically cleave gene mRNA transcripts encoding the chemorepellant. Ribozymes thus prevents translation of the target mRNA and prevents expression of the gene product. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage.

In another embodiment, the invention is a method of treating cancer in a patient suffering therefrom comprising inducing migration of an immune cell toward a cancer cell by inhibiting the activity of a chemorepellant released from a cancer cell. “Treating” or “treatment” includes preventing or delaying the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. A “patient” refers to a human subject in need of treatment.

In specific embodiments, the cancer is a solid tumor. In one embodiment, the solid tumor is selected from the group consisting of colon, prostate, breast, lung, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, brain, head and neck cancer. As used herein, a “therapeutically effective amount” in reference to inhibition of a chemorepellant is an amount sufficient to inhibit negative migration of an immune cell and ameliorate a disease or condition of a patient or achieve a desired outcome.

In reference to inducing chemotaxis, a “therapeutically effective amount” is an amount sufficient to induce negative migration of a migratory cell and ameliorate a disease or condition of a patient or achieve a desired outcome.

As used herein, “migratory cells” are those cells which are capable of movement from one place to another in response to a stimulus. Human migratory cells include those involved in the processes of cancer, immunity, angiogenesis or inflammation and also include those identified to play a role in other disease states or conditions. Migratory cells include, but are not limited to, immune cells, hematopoietic cells, neural cells, epithelial cells, mesenchymal cells, stem cells, germ cells and cells involved in angiogenesis.

Immune cells include, but are not limited to, monocytes, Natural Killer (NK) cells, dendritic cells (which could be immature or mature), subsets of dendritic cells including myeloid, plasmacytoid (also called lymphoid) or Langerhans; macrophages such as histiocytes, Kupffer's cells, alveolar macrophages or peritoneal macrophages; neutrophils, eosinophils, mast cells, basophils; B cells including plasma B cells, memory B cells, B-1 cells, B-2 cells; CD45RO (naive T), CD45RA (memory T); CD4 Helper T Cells including Th1, Th2 and Tr1/Th3; CD8 Cytotoxic T Cells, Regulatory T Cells and Gamma Delta T Cells.

Hematopoietic cells include, but are not limited to, pluripotent stem cells, multipotent progenitor cells and/or progenitor cells committed to specific hematopoietic lineages. The progenitor cells committed to specific hematopoietic lineages can be of T cell lineage, B cell lineage, dendritic cell lineage, neutrophil lineage, Langerhans cell lineage and/or lymphoid tissue-specific macrophage cell lineage. The hematopoietic cells can be derived from a tissue such as bone marrow, peripheral blood (including mobilized peripheral blood), umbilical cord blood, placental blood, fetal liver, embryonic cells (including embryonic stem cells), aortal-gonadal-mesonephros derived cells, and lymphoid soft tissue. Lymphoid soft tissue includes the thymus, spleen, liver, lymph node, skin, tonsil and Peyer's patches. In other embodiments, hematopoietic cells can be derived from in vitro cultures of any of the foregoing cells, and in particular in vitro cultures of progenitor cells.

Neural cells are cells of neural origin and include neurons and glia and/or cells of both central and peripheral nervous tissue.

Epithelial cells include cells of a tissue that covers and lines the free surfaces of the body. Such epithelial tissue includes cells of the skin and sensory organs, as well as the specialized cells lining the blood vessels, gastrointestinal tract, air passages, lungs, ducts of the kidneys and endocrine organs.

Mesenchymal cells include, but are not limited to, cells that express typical fibroblast markers such as collagen, vimentin and fibronectin.

Cells involved in angiogenesis are cells that are involved in blood vessel formation and include cells of endothelial origin and cells of mesenchymal origin.

Germ cells are cells specialized to produce haploid gametes.

In certain embodiment, the human migratory cell is an immune cell. In other embodiments, the immune cell is selected from the group consisting of lymphocytes, monocytes, neutrophils, eosinophils and mast cells. In a further embodiment, the immune cell is a neutrophil or an eosinophil.

As used herein, the terms “contact” or “contacting” means the act of touching or bringing together two entities or things in such proximity as will allow an influence of at least one on the other. The definition, while inclusive of physical contact is not so limited.

Based on their ability to induce negative chemotaxis, the chemorepellant proteins or biologically active fragments thereof as described herein are useful for inhibiting the induction of chemotaxis of migratory cells toward a chemotactic site. In one embodiment, the chemorepellant comprises a sequence that has substantial identity to the amino acid sequence of a protein selected from the proteins set forth in Tables 1 to 9, or to a biologically active fragment thereof. In some embodiment, the chemorepellant protein comprises a sequence that has substantial identity to a protein selected from the proteins set forth in Tables 10 to 11, or to a biologically active fragment thereof. In another embodiment, the protein comprises a sequence that has substantial identity to the sequence of a protein selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein, or to a biologically active fragment of any of thereof. As used herein, a “chemotactic site” is a site that induces positive chemotaxis of migratory cells. Chemotactic sites include sites of inflammation, medical implants, transplants and angiogenesis.

The chemorepellants described herein are useful for inhibiting the induction of chemotaxis of migratory cells toward a site of inflammation. Inhibiting migratory cell chemotaxis toward a site of inflammation can result in a reduction or amelioration of an inflammatory response in situations such as bacterial infection, tissue injury-induced inflammation (e.g., ischemia-reperfusion injury), complement-induced inflammation, oxidative stress (e.g., hemodialysis), immune complex-induced inflammation (e.g., antibody-mediated glomerunephritis), cytokine-induced inflammation (e.g., rheumatoid arthritis), antineutrophil cytoplasmic antibodies and vasculitis (e.g., autoimmunity against neutrophil components), genetic disorders of neutrophil regulations (e.g., hereditary periodic fever syndromes), implant related inflammation, and cystic fibrosis.

In certain embodiments, the invention is a method of treating an inflammatory condition in a patient suffering therefrom comprising administering to said patient a therapeutically effective amount of a chemorepellant described herein. In certain other embodiments, the invention is a method of treating an inflammatory condition in a patient suffering therefrom comprising administering to said patient a therapeutically effective amount of a chemorepellant described herein. Inflammatory conditions include, but are not limited to, appendicitis, peptic, gastric or duodenal ulcers, peritonitis, pancreatitis, acute or ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, inflammatory bowel disease (including, for example, Crohn's disease and ulcerative colitis), enteritis, Whipple's disease, asthma, chronic obstructive pulmonary disease, acute lung injury, ileus (including, for example, post-operative ileus), allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, pneumoultramicroscopic silicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virus, herpes, disseminated bacteremia, Dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatid cysts, burns, dermatitis, dermatomyositis, urticaria, acne, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease, celiac disease, congestive heart failure, adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillan-Barre syndrome, neuritis, neuralgia, uveitis, arthritides, arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcet's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, Type II diabetes, Retier's syndrome, Hodgkins disease and injection site reaction.

Injection site reaction is a term generally used to describe inflammation in and around a site of injection. Injection site reaction has been observed with the injection of numerous pharmaceutical agents including, but not limited, chemotherapeutic drugs, immunomodulator drugs, and vaccines. The present invention encompasses a method for the treatment or reduction of injection site reaction comprising administration of a chemorepellant described herein to the injection site. The chemorepellant can, for example, be administered before, during or after injection. In some embodiments, exenatide or analog thereof can be administered topically at the site of the injection.

In another embodiment, the invention is a method of inhibiting positive chemotaxis toward a medical implant. The medical implant can be contacted or coated with a chemorepellant described herein. The proteins can also be administered locally at the site of the medical implant. A medical implant is defined as a device or entity implanted into a surgically or naturally formed cavity of the body. Medical implants include, but are not limited to, stents, pacemakers, pacemaker leads, defibrillators, drug delivery devices, sensors, pumps, embolization coils, sutures, electrodes, cardiovascular implants, arterial stents, heart valves, orthopedic implants, dental implants, bone screws, plates, catheters, cannulas, plugs, fillers, constrictors, sheets, bone anchors, plates, rods, seeds, tubes, or portions thereof. In addition to the chemorepellant, the medical implant can be coated with a cell-growth potentiating agent, an anti-infective agent and/or an anti-inflammatory agent.

In yet another embodiment, the invention is a method of inhibiting positive chemotaxis toward an organ transplant or tissue graft. Organ transplants and tissue grants include, but are not limited to, renal, pancreatic, hepatic, lymphoid and cardiac grafts and organs. Lymphoid grafts include a splenic graft, a lymph node derived graft, a Peyer's patch derived graft, a thymic graft and a bone marrow derived graft. In an additional embodiment, the invention is a method of treating a patient suffering from transplant or graft rejection comprising administering an inventive chemorepellant.

As discussed above, the inventive chemorepellants can be used to inhibit chemotaxis toward a site of angiogenesis. A site of angiogenesis is a site where blood vessels are being formed. In one embodiment, the invention is a method of inducing negative chemotaxis of endothelial cells away from a site of angiogenesis. The invention also encompasses a method of inhibiting angiogenesis in a patient in need thereof comprising administering an inventive chemorepellant In a further embodiment, the invention is a method of treating cancer or a tumor comprising administering an inventive chemorepellant in an amount effective to inhibit angiogenesis. According to another aspect of the invention, a method of inhibiting endothelial cell migration to a tumor site in a subject is provided. The method involves locally administering to or contacting an area surrounding a tumor site in need of such treatment an inventive chemorepellant in an amount effective to inhibit endothelial cell migration into the tumor site in the subject.

Exemplary cancers and tumors that can be treated according to the methods of the invention include, for example, biliary tract cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer, gastric cancer; hematological neoplasms, including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer (hepatocarcinoma); lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma, basocellular cancer and squamous cell cancer; testicular cancer, including germinal tumors (seminoma, non-seminoma [teratomas, choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor.

The invention also encompasses a method of contraception in a patient in need thereof comprising administering an inventive chemorepellant in an amount effective to inhibit migration of germ cells in the subject. According to another aspect of the invention, a method of treating infertililty and premature labor is provided. The method comprises administering a compound described above in an amount effective to inhibit immune cells from migrating close to a germ cell in the subject.

The treatment methods disclosed herein involve administering, either locally or systemically, to a selected site in a subject in need of such a treatment a chemorepellant of the invention in an amount effective to induce negative chemotaxis of a human migratory cell or an inhibitor of a chemorepellant in an amount effect to suppress negative chemotaxis of an immune cell. For example, a “therapeutically effective amount” in reference to the treatment of an inflammatory condition encompasses an amount sufficient to induce negative chemotaxis of an immune cell and/or ameliorate a symptom of the inflammatory condition.

In certain embodiments, the chemorepellant can be co-administered with a second agent (e.g., another chemoattractant or with any drug or agent which is not itself a chemoattractant). Co-administered agents, compounds, chemoattractants or therapeutics need not be administered at exactly the same time. In certain embodiments, however, the chemorepellant is administered substantially simultaneously as the second agent. By “substantially simultaneously,” it is meant that the chemorepellant is administered before, at the same time, and/or after the administration of the second agent. Second agents include, for example, anti-inflammatory agents, anti-cancer agents, anti-infective agents, immune therapeutics (immunosuppresants) and other therapeutic compounds. A second agent can be chosen based on the condition or disease to be treated. For example, in a method of treating cancer or a tumor, the chemorepellant can be administered with an anti-cancer agent. Similarly, in a method of treating an inflammatory condition, the chemorepellant can be administered with an anti-inflammatory agent, an anti-infective agent or an immunosuppressant.

An anti-infective agent is an agent which reduces the activity of or kills a microorganism and includes: Aztreonam; Chlorhexidine Gluconate; Imidurea; Lycetamine; Nibroxane; Pirazmonam Sodium; Propionic Acid; Pyrithione Sodium; Sanguinarium Chloride; Tigemonam Dicholine; Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride; Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium; Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; Biphenamine Hydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate; Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; Carbenicillin Indanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium; Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate; Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride; Cefetecol; Cefixime; Cefinenoxime Hydrochloride; Cefmetazole; Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium; Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine; Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil; Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; Cephalexin Hydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol; Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol Pantothenate Complex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate; Chloroxylenol; Chlortetracycline Bisulfate; Chlortetracycline Hydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride; Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin; Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride; Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; Cloxacillin Sodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin; Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone; Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline; Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline; Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; Droxacin Sodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride; Erythromycin; Erythromycin Acistrate; Erythromycin Estolate; Erythromycin Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Lactobionate; Erythromycin Propionate; Erythromycin Stearate; Ethambutol Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine; Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin; Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid; Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin; Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole; Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin; Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride; Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; Meclocycline Sulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem; Methacycline; Methacycline Hydrochloride; Methenamine; Methenamine Hippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim; Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin lydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; Nalidixate Sodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate; Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone; Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium; Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin; Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin G Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V; Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V Potassium; Pentizidone Sodium; Phenyl Aminosalicylate; Piperacillin Sodium; Pirbenicillin Sodium; Piridicillin Sodium; Pirlimycin Hydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate; Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin; Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; Rosaramicin Butyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate; Rosaramicin Stearate; Rosoxacil; Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin; Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride; Spiramycin; Stallimycin Hydrochloride; Steffimycin; Streptomycin Sulfate; Streptonicozid; Sulfabenz: Sulfabenzamide; Sulfacetamide; Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium; Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine; Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole; Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole; Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; Suncillin Sodium; Talampicillin Hydrochloride; Teicoplanin; Temafloxacin Hydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride; Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium; Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin; Zorbamycin; Difloxacin Hydrochloride; Lauryl Isoquinolinium Bromide; Moxalactam Disodium; Ornidazole; Pentisomicin; and Sarafloxacin Hydrochloride.

Exemplary anti-cancer agents include Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexatc; Eflorithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Fluorocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b; Iproplatini; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Podofilox; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxotere; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporlin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate Virlrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; and Zorubicin Hydrochloride.

Exemplary immunosuppressants include Azathioprine; Azathioprine Sodium; Cyclosporine; Daltroban; Gusperimus Trihydrochloride; Sirolimus; and Tacrolimus. Exemplary anti-inflammatory agents include Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; and Zomepirac Sodium.

As used herein, “treatment” and/or “treating” refer to therapeutic treatment as well as prophylactic treatment or preventative measures. The chemorepellant and/or other therapeutic (such as an antibody to the chemorepellant) can be administered in pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient. The excipient can be chosen based on the expected route of administration of the composition in therapeutic applications. The route of administration of the composition depends on the condition to be treated. Routes of administration include, but are not limited to, parenteral, topic, oral, intramuscular, intravenous administration. The route of administration and the dosage of the composition to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. In one embodiment, the chemorepellant or a composition thereof is administered locally.

The therapeutic compositions used in the inventive methods can be administered parenterally such as, for example, by intravenous, intramuscular, intrathecal, or subcutaneous injection. Parenteral administration can be accomplished by incorporating the therapeutic compositions of the present invention into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents. Parenteral formulations may also include antibacterial agents such as, for example, benzyl alcohol or methyl parabens, antioxidants such as, for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes, or multiple dose vials made of glass or plastic.

The invention is illustrated by the following examples which are not meant to be limiting in any way.

EXEMPLIFICATION Example 1 Identification of Modulators of Cell Migration Present in Tumor Environments

Objective: To identify the agents present in tumor microenvironments that have the ability to modulate the migration of immune cell subsets.

Materials and Methods:

Cystic fluid samples: Fluids from ovarian carcinoma patients were collected during surgical procedures under a signed informed consent. Fluids were centrifuged to remove the debris. The supernatants were supplemented with cocktail of protease inhibitors and divided into aliquots and stored at −80° C. till further processing. Samples were evaluated to study their effects on migration of neutrophils in transwell migration assays in Boyden chambers for their chemoattraction (CA) and chemorepulsion (CR) activities as described below.

Chromatographic separation: Cystic fluid (0.2 ml at 65 mg/ml) was loaded on a Superdex 200 10/300 GL column (GE Healthcare) and fractionated at the rate of 0.5 ml/min. Fractions (1 ml) were collected in tubes preloaded with 10 μl of 100× concentration Complete EDTA-free Protease Inhibitor Cocktail (Roche). These fractions were evaluated for CA CR activities in transwell migration assays described below.

One and two dimensional SDS-PAGE analysis: Fractions collected from S-200 chromatography with CR activity and the adjacent fractions without CR activity were further fractionated by one and two dimensional SDS-PAGE. Proteins band and/or spots differentially present in S-200 fractions with CR activity were excised manually, digested with trypsin, and subjected to either LC-MS/MS (1-D bands) or MALDI (2-D spots) analysis.

The chemorepulsive activity of the cystic fluid, fractions collected from S-200 chromatography and the proteins listed below was determined as follows:

Prior to beginning the assay, the following were prepared:

0.5% Fetal Calf Serum (FCS) in Iscove's Modified Dulbecco's Medium (IMDM) (Assay Medium) (Both from ATCC). Migratory cells at a concentration of 2×10⁷ cells/ml in Assay Medium. Four serial (3-fold) dilutions of the ligand of interest in Assay Medium. The assay plates are Neuroprobe ChemoTx plates, part number 206-3 (3 um pore size) for neutrophils. 31 μl of the following solutions were pipetted into each well: For media controls and for chemorepulsion samples, Assay Medium was used. For chemoattraction samples, appropriate dilution of ligand was used. The membrane was carefully placed onto the plate, starting at one side and then slowly lowering the other edge onto the plate. 29 μl of the following were pipetted onto the top of each circle: For media controls and chemoattraction samples, use Assay Medium. For chemorepulsion samples, use the appropriate dilution of ligand. 2 μl of cells (40,000 cells) were added to each bubble of liquid from step 7.

The plate was covered with the supplied lid and incubated for the desired time at 37° C. in 5% CO₂. Unless otherwise indicated, the incubation time was 1 hour for neutrophils and 3 hours for T cells. For monocytes and B cells, the incubation time was 2 hours.

After the desired assay time, the liquid was removed from the top of the plate using a Kimwipe.

The membrane was carefully removed from the top of the plate and discarded. The plate was examined under a microscope to look for ligand crystallization, contamination and overall migration.

White read plates were preloaded with 25 ul PBS. Using a multichannel pipettor, 5 ul of Cell Titer Glo (Promega # G7572) was added to each well. Using a multichannel pipettor set at 30 ul, lysed cell solution was transferred to white read plates pre-loaded with PBS. The plate was read using the BioTek Synergy4 plate reader in order to quantify the number of migrated cells.

Results:

From mass spectrometry (MS) analysis, 86 proteins in the chemorepulsion active chromatography fraction have been identified which are represented in the following table.

TABLE 1 Proteins present in specific active fragments of S200 Identified Proteins Accession Number A1BG Alpha-1B-glycoprotein IPI00022895 precursor A2M Alpha-2-macroglobulin IPI00478003 precursor ACTA2 Actin, aortic smooth IPI00008603 (+9) muscle ACTB Actin, cytoplasmic 1 IPI00021439 (+2) AFM Afamin precursor IPI00019943 AHSG Alpha-2-HS-glycoprotein IPI00022431 (+1) precursor ALB Isoform 1 of Serum albumin IPI00745872 (+1) precursor Alpha 2 HS-glycoprotein P02765; gi: 112910 ANPEP Aminopeptidase N IPI00221224 APOA1 Apolipoprotein A-I IPI00021841 (+1) precursor apolipoprotein A-1 P02647 apolipoprotein A-IV P06727 C1RL Complement C1r IPI00009793 (+2) subcomponent-like protein precursor C2 Complement C2 precursor IPI00303963 (Fragment) C3 Complement C3 precursor IPI00783987 (Fragment) C4A Complement component 4A IPI00643525 C9 Complement component C9 IPI00022395 precursor carbonic anhydrase 1 P00915 CD163 Isoform 1 of Scavenger IPI00104074 (+3) receptor cysteine-rich type 1 protein M130 precursor CFB Isoform 1 of Complement IPI00019591 factor B precursor (Fragment) CP Ceruloplasmin precursor IPI00017601 EEF1A2 Elongation factor 1-alpha 2 IPI00014424 (+3) F2 Prothrombin precursor IPI00019568 (Fragment) GC Vitamin D-binding protein IPI00555812 (+1) precursor GSN Isoform 1 of Gelsolin IPI00026314 (+1) precursor H2AFV Histone H2AV IPI00018278 (+15) HABP2 Hyaluronan-binding IPI00746623 protein 2 precursor HBA2; HBA1 Hemoglobin subunit IPI00410714 (+1) alpha HBB Hemoglobin subunit beta IPI00654755 (+1) hemoglobin beta P68871 hemopexin P02790 HIST1H1D Histone H1.3 IPI00217466 (+2) HIST1H2AM; HIST1H2AG; HIST1H2AJ; IPI00291764 (+9) HIST1H2AL; HIST1H2AK; HIST1H2AI Histone H2A type 1 HIST2H3A; HIST2H3C; HIST2H3D IPI00171611 (+7) Histone H3.2 HIST2H4A; HIST1H4C; HIST1H4A; IPI00453473 HIST1H4I; HIST1H4E; HIST1H4F; HIST1H4K; HIST1H4H; HIST4H4; HIST1H4L; HIST1H4D; HIST1H4J; HIST2H4B; HIST1H4B Histone H4 HPX Hemopexin precursor IPI00022488 HRG Histidine-rich glycoprotein IPI00022371 precursor HRNR Hornerin IPI00398625 (+2) IGFALS Insulin-like growth IPI00020996 factor-binding protein complex acid labile chain precursor IGHD IGHD protein IPI00418422 (+2) IGHG1 IGHG1 protein IPI00448925 IGHG1 IGHG1 protein IPI00815926 IGHG3 IGHG3 protein IPI00472345 IGHM; IGH@ IGHM protein IPI00472610 IGHV1OR15-1 Ig heavy chain V-I IPI00009792 region V35 precursor IGHV3OR16-13; IGHA1 IGHA1 IPI00061977 protein IGHV3OR16-13; IGHA1 IGHA1 IPI00430842 protein IGHV4-31 IGHV4-31 protein IPI00784822 IGKV1-5 IGKV1-5 protein IPI00419424 (+19) IGL@ IGL@ protein IPI00154742 ITIH2 Inter-alpha-trypsin inhibitor IPI00305461 (+1) heavy chain H2 precursor ITIH4 Isoform 1 of Inter-alpha- IPI00294193 trypsin inhibitor heavy chain H4 precursor ITIH4 Isoform 2 of Inter-alpha- IPI00218192 (+3) trypsin inhibitor heavy chain H4 precursor KNG1 Isoform LMW of IPI00215894 (+1) Kininogen-1 precursor KPRP Keratinocyte proline-rich IPI00514908 protein KRT1 Keratin, type II cytoskeletal 1 IPI00220327 (+1) KRT10 Keratin, type I cytoskeletal IPI00009865 (+1) 10 KRT14 Keratin, type I IPI00384444 cytoskeletal 14 KRT16 Keratin, type I IPI00217963 cytoskeletal 16 KRT2 Keratin, type II cytoskeletal IPI00021304 (+1) 2 epidermal KRT5 Keratin, type II cytoskeletal 5 IPI00009867 KRT6A Keratin, type II IPI00300725 cytoskeletal 6A KRT9 Keratin, type I cytoskeletal 9 IPI00019359 (+1) LDHA Isoform 1 of L-lactate IPI00217966 (+2) dehydrogenase A chain LUM Lumican precursor IPI00020986 (+1) LYZ Lysozyme C precursor IPI00019038 (+1) plasma retinol-binding protein P02753 SERPINA1 Isoform 1 of Alpha-1- IPI00553177 antitrypsin precursor SERPINA3 Alpha-1- IPI00550991 (+1) antichymotrypsin precursor SERPINA7 Thyroxine-binding IPI00292946 globulin precursor SERPIND1 Serpin peptidase IPI00292950 (+1) inhibitor, clade D (Heparin cofactor), member 1 SERPINF2 SERPINF2 protein IPI00029863 (+1) SLPI Antileukoproteinase IPI00008580 precursor sp_ALBU_BOVIN IPIsp_ALBU_BOVIN sp_ANT3_HUMAN IPIsp_ANT3_HUMAN sp_TRYP_PIG IPIsp_TRYP_PIG TF Serotransferrin precursor IPI00022463 (+2) transthyretin P02766 Putative uncharacterized protein IPI00426051 DKFZp686C15213 cDNA FLJ78387 IPI00876888 Ig heavy chain V-III region CAM IPI00382482 Single-chain Fv (Fragment) IPI00470652 uncharacterized protein IPI00735451 ENSP00000375035 uncharacterized protein IPI00829845 ENSP00000375026 YWHAZ 14-3-3 protein zeta/delta IPI00021263 (+1) zinc-alpha-2-glycoprotein P25311

Some of these proteins were evaluated individually and in combinations for their effects on CA and CR activity. Of these proteins, actin, 14-3-3 zeta/delta, apolipoprotein A1 and hemopexin showed the greatest CA and/or CR activities. FIGS. 1 through 6 represent the effect of whole cyst fluid, Superdex 200 fractions, Actin and 14-3-3 individually, the same two proteins assayed in combination, Apolipoprotein A1, and hemopexin on migration of human neutrophils in CA and CR modes.

Legends for the Figures:

FIG. 1: Effect of Cystic fluid on migration of human neutrophils. Human neutrophils were tested at different concentrations of cyst fluid: neat (undiluted), and at 1:3, 1:10 and 1:30 diluted in media. Both chemoattraction (CA) and chemorepulsion were measured using a Boyden chamber transwell migration assay. Cystic fluid has efficiently repelled human neutrophils as studied by transwell migration assays at all concentrations tested.

FIG. 2: Evaluation of S-200 chromatography fractionation of cystic fluids on human neutrophils in transwell migration assay. Fractions were evaluated for chemoattraction (CA) and chemorepulsion of human neutrophils using a Boyden chamber transwell migration assay. Fractions A15 and B1 have the highest neutrophil repulsive activities as compared to other fractions.

FIG. 3: Effect of human actin and 14-3-3 on migration of human neutrophils. Actin and 14-3-3 were evaluated at different concentrations for their abilities to induce chemorepulsion (CR) of human neutrophils using a Boyden chamber transwell migration assay. Human neutrophils were effectively repelled by actin in transwell migration assays.

FIG. 4: Effect of 1:1 combination of Actin and 14-3-3 on migration of human neutrophils. Actin and 14-3-3 were evaluated in 1:1 combination at different concentrations for their ability to induce chemoattraction (CA) and chemorepulsion (CR) of human neutrophils using a Boyden chamber transwell migration assay. Actin and 14-3-3 in combination effectively modulated human neutrophil migrations in transwell migration assays.

FIG. 5: Effect of apolipoprotein A1 on migration of human neutrophils. Apolipoprotein A1 was evaluated at different concentrations for its ability to induce chemoattraction (CA) and chemorepulsion (CR) of human neutrophils using a Boyden chamber transwell migration assay. Human neutrophils were effectively repelled by apolipoprotein A1 at 5.1 microM concentration.

FIG. 6: Effect of hemopexin on migration of human neutrophils. Hemopexin was evaluated at different concentrations for its ability to induce chemoattraction (CA) and chemorepulsion (CR) of human neutrophils using a Boyden chamber transwell migration assay. Human neutrophils were effectively attracted at 8.8 microM concentration of hemopexin.

Example 2 Identification of Modulators of Cell Migration Present in Mammalian Cancer Cell Line Supernatants

Objective: To identify the agents present in mammalian cancer cell lines that have the ability to modulate the migration of immune cell subsets.

Materials and Methods: Mammalian Cancer Cell Lines:

Cancer cell lines were cultured in serum containing media until desired confluence is reached. Culture conditions were switched to serum-free media and supernatants collected everyday up to certain number of days. The supernatants were supplemented with cocktail of protease inhibitors and divided into aliquots and stored at −80 C until further processing. Depending on the volume of culture supernatant, they were either concentrated 10 times or evaluated unconcentrated to study their effects on neutrophil migration Boyden chamber transwell migration assays.

Chromatographic Separation:

Supernatants were further concentrated and loaded on a Superdex 200 10/300 GL column (GE Healthcare) and fractionated at the rate of 0.5 ml/min. Fractions (1 ml) were collected in tubes preloaded with 10 ul of 100× concentration Complete EDTA-free Protease Inhibitor Cocktail (Roche). These fractions were evaluated for chemoattraction (CA) and chemorepulsion (CR) activities in transwell migration assays described below.

Supernatants for the breast cancer cell line, SK-BR-3 were first dialyzed overnight and then loaded on a HiTrap-Q Fast Flow anion exchange column and fractionated at a rate of 1 mL/min. 3 mL fractions were desalted and evaluated for chemoattraction (CA) and chemorepulsion (CR) activities in transwell migration assays as described below.

One Dimensional SDS-PAGE Analysis:

Fractions collected from S-200 and anion exchange chromatography with CR activity and the adjacent fractions without CR activity were further fractionated by one dimensional SDS-PAGE. Proteins bands differentially present in S-200 fractions with CR activity were excised manually, digested with trypsin, and subjected to LC-MS/MS.

The chemorepulsive activity of the supernatants, fractions collected from S-200 and anion exchange chromatography and the proteins listed below were determined as follows:

Transwell migration Assay: 1. Prior to beginning the assay, the following were prepared: a. 0.5% Fetal Calf Serum (FCS) in Iscove's Modified Dulbecco's Medium (IMDM) (Assay Medium) (Both from ATCC). b. Migratory cells at a concentration of 2×10₇ cells/ml in Assay Medium. 2. The assay plates are Neuroprobe ChemoTx plates, part number 206-3 (3 um pore size) for neutrophils. 3. 31 μl of the following solutions were pipetted into each well: a. For media controls and for chemorepulsion samples, Assay Medium was used. b. For chemoattraction samples, appropriate dilution of ligand was used. 4. The membrane was carefully placed onto the plate, starting at one side and then slowly lowering the other edge onto the plate. 5. 29 μl of the following were pipetted onto the top of each circle: a. For media controls and chemoattraction samples, Assay Medium was used. b. For chemorepulsion samples, the appropriate dilution of ligand was used. 6. 2 μl of cells (40,000 cells) were added to each bubble of liquid from step 7. 7. The plate was covered with the supplied lid and incubated for 1 hour at 37° C. in 5% CO₂. 8. The liquid was then removed from the top of the plate using a Kimwipe. 9. The plate was then examined under a microscope to look for crystallization, contamination and overall migration. From this point assay plates were either processed by method A: CTG (Cell Titer Glo via relative luminescence units for read out) or method B: Guava (via cell count for read out).

Method A:

1. White read plates were preloaded with 25 ul PBS and 5 ul of Cell Titer Glo (Promega #G7572) was added to each well of the transmigration plate. 2. Using a multichannel pipettor set at 30 ul, lysed cell solution was transferred to white read plates pre-loaded with PBS. 3. The plate was read using the BioTek Synergy4 plate reader in order to quantify the number of migrated cells.

Method B:

U-bottom 96 well plates were preloaded with 50 ul assay media and the contents of the Neuroprobe plates were transferred to the U-bottom plate. Equal volumes of Guava viacount reagent was added to each well to stain the cells. The plate was then incubated for 5 minutes in the dark at room temperature. 1% paraformaldehyde was added to fix the cells and they were then sealed with adhesive film and stored at 4° C. overnight. The Guava Easy Cyte Plus was used to read the plate and quantify the number of migrated cells.

Bands from supernatant fractions that exhibited chemorepulsive activity were sent out for MS (Liquid chromatography/Mass Spectrometry/Mass Spectrometry) analysis (outsourced). Commercially available proteins corresponding to proteins identified in Mass Spectrometry were then tested in cell migration assay.

Protein identification was performed by outside sources using nano LC/MS/MS (Liquid Chromatography/Mass Spectrometry/Mass Spectrometry) on an LTQ (“linear trap quadrupole”) mass spectrometer. Protein samples were submitted in a gel or solution and were first digested robotically using trypsin to create a peptide mixture (alternate enzymes may be employed if necessary). Peptides were then injected on a custom-designed LC column set-up and eluted into the mass spectrometer where MS and MS/MS were performed. Product ion data was searched using forward and reversed database searching methods to allow assessment of false discovery rates and ensure only correct protein identifications were reported. Search results were parsed into the SCAFFOLD™ visualization software to allow further validation of protein assignments through the PROTEINPROPHET™ and PEPTIDEPROPHET™¹ tools.

The methods used for In-gel digestion are as below:

Samples were subjected to proteolytic digestion on a ProGest workstation as follows:

The samples were reduced with DTT at 60° C., allowed to cool to room temperature, alkylated with iodoacetamide, incubated at 37° C. for 4 h in the presence trypsin and formic acid was added to stop the reaction.

The method used for Mass Spectrometry—Solution Based are below:

Samples were subjected to C18 capture using ZipTips. They were aspirated across equilibrated C18 ZipTip, washed in 0.1% formic acid, eluted in 80% acetonitrile in 0.1% formic acid, concentrated by vacuum centrifugation and resuspended in 0.1% formic acid for injection.

The methods used for LC/MS/MS (data-dependent) are as below:

Samples were analyzed by nano LC/MS/MS on a ThermoFisher LTQ XL or Orbitrap XL. 30 μl of hydrolysate were loaded on a 75 μm C12 vented column at a flow-rate of 10 μL/min and eluted at 300 nL/min and a 1 h gradient was employed. MS/MS data were searched using a local copy of Mascot (www.matrixscience.com) The parameters for all LC/MS/MS (Mascot) searches were as follows: Type of search: MS/MS Ion Search

Enzyme: Trypsin

Fixed modifications: Carbamidomethyl (C) Variable modifications: Oxidation (M, Acetyl (N-term, Pyro-glu (N-term Q) Mass values: Monoisotopic

Protein Mass Unrestricted Peptide Mass Tolerance: ±10 ppm (Orbitrap); ±2.0 Da (LTQ) Fragment Mass Tolerance: ±0.5 Da (LTQ) Max Missed Cleavages: 1

Samples were processed in the SCAFFOLD™ Algorithm (www.proteomesoftware.com) using .DAT files generated by MASCOT™. Parameters for LTQ data require a minimum of 3 peptides matching per protein with minimum probabilities of 95% at the protein level and 50-80% at the corresponding peptide level. QTOF/Orbitrap data require a minimum of 2 peptides with the same minimum probability thresholds due to the superior mass accuracy of that instrument.

NOTE: Detailed protocols for each of these methods can be found in the technical information section of http://www.prsproteomics.com.

NOTE: SK-BR-3 was outsourced using LC/MS/MS performed at University of Georgia, Proteomics Resource Facility.

Results:

The chemorepulsive activity of supernatants, fractions collected from chromatography and commercially available proteins are shown in FIGS. 7-39.

Proteins identified in the chemorepulsive supernatant fractions by LC/MS/MS (mass spectrometry) are shown in the Tables below:

TABLE 2 Proteins identified by MS in Renal Cell Lines ACHN and 786-O Protein: Accession # ACBD3 Golgi resident protein GCP60 IPI00009315 ADPRHL2 Poly(ADP-ribose) glycohydrolase ARH3 IPI00015865 AK2 Isoform 1 of Adenylate kinase isoenzyme 2, mitochondrial IPI00215901 (+1) AKR1A1 Alcohol dehydrogenase IPI00220271 AKR1B1 Aldose reductase IPI00413641 AKR1B10 Aldo-keto reductase family 1 member B10 IPI00105407 AKR1C1 Aldo-keto reductase family 1 member C1 IPI00029733 AKR1C2 Aldo-keto reductase family 1 member C2 IPI00005668 AKR1C3 Aldo-keto reductase family 1 member C3 IPI00291483 (+1) ANP32B Isoform 1 of Acidic leucine-rich nuclear phosphoprotein 32 family IPI00007423 (+1) member B ANXA1 Annexin A1 IPI00218918 ANXA2 Annexin A2 IPI00455315 APEX1 DNA-(apurinic or apyrimidinic site) lyase IPI00215911 APOA1BP Isoform 1 of Apolipoprotein A-I-binding protein precursor IPI00168479 (+1) ARHGDIA Rho GDP-dissociation inhibitor 1 IPI00003815 (+1) ARMET Protein ARMET precursor IPI00328748 ASF1A Histone chaperone ASF1A IPI00292168 BSG Isoform 2 of Basigin precursor IPI00019906 (+1) C11orf54 Isoform 3 of Ester hydrolase C11orf54 IPI00061507 (+2) C19orf33 Isoform 1 of Immortalization up-regulated protein IPI00030767 C1orf128 Isoform 1 of UPF0424 protein C1orf128 IPI00015351 C7orf24 Uncharacterized protein C7orf24 IPI00031564 CA12 Isoform 1 of Carbonic anhydrase 12 precursor IPI00012895 (+1) CA2 Carbonic anhydrase 2 IPI00218414 (+1) CAB39 Calcium-binding protein 39 IPI00032561 CALD1 Isoform 4 of Caldesmon IPI00218696 CALM2; CALM1; CALM3 Calmodulin IPI00075248 (+2) CAPG Macrophage-capping protein IPI00027341 (+1) CAPZA2 F-actin-capping protein subunit alpha-2 IPI00026182 (+3) CASP3 Caspase-3 precursor IPI00292140 CAST Isoform 2 of Calpastatin IPI00220857 (+11) CCDC25 Coiled-coil domain-containing protein 25 IPI00396174 (+1) CDH13 Cadherin-13 precursor IPI00024046 (+2) CDV3 Isoform 1 of Protein CDV3 homolog IPI00014197 (+2) GSTM3 Glutathione S-transferase Mu 3 IPI00246975 GSTP1 Glutathione S-transferase P IPI00219757 (+1) GUK1 Guanylate kinase IPI00182293 (+3) HDDC2 Isoform 2 of HD domain-containing protein 2 IPI00386751 (+1) HDGF Hepatoma-derived growth factor IPI00020956 HDHD1A Haloacid dehalogenase-like hydrolase domain containing protein IPI00302436 HDHD3 Haloacid dehalogenase-like hydrolase domain-containing protein 3 IPI00009931 HLA-B; HLA-A; HLA-C; LOC441528; XXbac- IPI00472676 (+2) BPG181B23.1; LOC728687; MICA; LOC100133382 HLA class I histocompatibility antigen, B-42 alpha chain precursor HMGA1 Isoform HMG-I of High mobility group protein HMG-I/HMG-Y IPI00179700 HMGB3 High mobility group protein B3 IPI00217477 (+2) HMGN1 Non-histone chromosomal protein HMG-14 IPI00554761 HN1 Isoform 1 of Hematological and neurological expressed 1 protein IPI00007764 (+1) HNRNPA2B1 Isoform B1 of Heterogeneous nuclear ribonucleoproteins A2/B1 IPI00396378 HPRT1 Hypoxanthine-guanine phosphoribosyltransferase IPI00218493 IAH1 Isoamyl acetate-hydrolyzing esterase 1 homolog IPI00419194 (+1) IGFBP7 Insulin-like growth factor-binding protein 7 precursor IPI00016915 IGSF8 Isoform 1 of Immunoglobulin superfamily member 8 precursor IPI00056478 (+1) IL6 Interleukin-6 precursor IPI00007793 (+2) ITIH5 inter-alpha trypsin inhibitor heavy chain precursor 5 isoform 1 IPI00328829 (+1) KIAA0174 Isoform 1 of Uncharacterized protein KIAA0174 IPI00024660 (+2) LASP1 Isoform 1 of LIM and SH3 domain protein 1 IPI00000861 (+2) LDHB L-lactate dehydrogenase B chain IPI00219217 LMAN2 Vesicular integral-membrane protein VIP36 precursor IPI00009950 LMNA Isoform A of Lamin-A/C IPI00021405 (+4) LMNB1 Lamin-B1 IPI00217975 LMNB2 Lamin-B2 IPI00009771 (+1) LOC100130561; HMG1L10 High mobility group protein 1-like 10 IPI00018755 (+3) M6PRBP1 Isoform A of Mannose-6-phosphate receptor-binding protein 1 IPI00106668 (+1) MAP1B Microtubule-associated protein 1B IPI00008868 MAPRE1 Microtubule-associated protein RP/EB family member 1 IPI00017596 MCM3 DNA replication licensing factor MCM3 IPI00013214 MDH1 Malate dehydrogenase, cytoplasmic IPI00291005 MDH2 Malate dehydrogenase, mitochondrial precursor IPI00291006 MMP14 Matrix metalloproteinase-14 precursor IPI00218398 (+1) NENF Neudesin precursor IPI00002525 NIPSNAP3A Protein NipSnap homolog 3A IPI00004845 (+1) NME2 Nucleoside diphosphate kinase IPI00604590 (+1) NPC2 Epididymal secretory protein E1 precursor IPI00301579 NPM1 Isoform 2 of Nucleophosmin IPI00220740 (+2) NQO2 Ribosyldihydronicotinamide dehydrogenase IPI00219129 (+3) NUDT1 Isoform p26 of 7,8-dihydro-8-oxoguanine triphosphatase IPI00004392 (+4) PARK7 Protein DJ-1 IPI00298547 PDAP1 28 kDa heat- and acid-stable phosphoprotein IPI00013297 PDIA6 Isoform 2 of Protein disulfide-isomerase A6 precursor IPI00299571 (+1) PEBP1 Phosphatidylethanolamine-binding protein 1 IPI00219446 PGLS 6-phosphogluconolactonase IPI00029997 PIR Pirin IPI00012575 PNPO Pyridoxine-5′-phosphate oxidase IPI00018272 (+1) POLDIP2 Polymerase delta-interacting protein 2 IPI00165506 POLR2H DNA-directed RNA polymerases I, II, and III subunit RPABC3 IPI00003309 PPIA Peptidyl-prolyl cis-trans isomerase A IPI00419585 (+4) PPIB peptidylprolyl isomerase B precursor IPI00646304 PPIF Peptidyl-prolyl cis-trans isomerase, mitochondrial precursor IPI00026519 PPP1R14C Protein phosphatase 1 regulatory subunit 14C IPI00290397 PRDX1 Peroxiredoxin-1 IPI00000874 (+1) PRDX2 Peroxiredoxin-2 IPI00027350 PRDX3 Thioredoxin-dependent peroxide reductase, mitochondrial precursor IPI00024919 (+1) PRDX6 Peroxiredoxin-6 IPI00220301 PROCR Endothelial protein C receptor precursor IPI00009276 PSPH Phosphoserine phosphatase IPI00019178 PTGDS Prostaglandin-H2 D-isomerase precursor IPI00013179 (+2) PTGR1 NADP-dependent leukotriene B4 12-hydroxydehydrogenase IPI00292657 PTMS Parathymosin IPI00550020 QDPR Dihydropteridine reductase IPI00014439 RAB11B Ras-related protein Rab-11B IPI00020436 (+2) RAB1A Isoform 1 of Ras-related protein Rab-1A IPI00005719 (+6) RAB5C Ras-related protein Rab-5C IPI00016339 RAD23A UV excision repair protein RAD23 homolog A IPI00008219 RALA Ras-related protein Ral-A precursor IPI00217519 (+1) RBM8A Isoform 1 of RNA-binding protein 8A IPI00001757 (+1) REXO2 Isoform 1 of Oligoribonuclease, mitochondrial precursor (Fragment) IPI00032830 (+1) RNASET2 Isoform 1 of Ribonuclease T2 precursor IPI00414896 (+1) RPE Isoform 1 of Ribulose-phosphate 3-epimerase IPI00335280 (+1) RPIA Ribose-5-phosphate isomerase IPI00026513 (+1) SAMD9 Isoform 1 of Sterile alpha motif domain-containing protein 9 IPI00217018 S100A11 Protein S100-A11 IPI00013895 S100A6 Protein S100-A6 IPI00027463 SCYE1 Multisynthetase complex auxiliary component p43 IPI00006252 (+1) SERPINB6 Putative uncharacterized protein DKFZp686I04222 IPI00413451 (+1) SMAP1 Isoform 1 of Stromal membrane-associated protein 1 IPI00102096 (+2) SNX12 Isoform 1 of Sorting nexin-12 IPI00438170 (+2) SOD1 Superoxide dismutase IPI00218733 (+1) SOD2 Superoxide dismutase [Mn], mitochondrial precursor IPI00022314 (+2) sp_TRYP_PIG IPIsp_TRYP_PIG SPINT2 Kunitz-type protease inhibitor 2 precursor IPI00011662 STX7 Isoform 1 of Syntaxin-7 IPI00289876 (+1) SUB1 Activated RNA polymerase II transcriptional coactivator p15 IPI00221222 TAGLN2 Transgelin-2 IPI00550363 TALDO1 Transaldolase IPI00744692 THOC4 THO complex subunit 4 IPI00328840 TP53I3 Isoform 1 of Putative quinone oxidoreductase IPI00384643 TPI1 Isoform 1 of Triosephosphate isomerase IPI00465028 TPK1 Thiamin pyrophosphokinase 1 IPI00072523 (+1) TPT1 Translationally-controlled tumor protein IPI00550900 TRIOBP TRIO and F-actin binding protein isoform 1 IPI00148768 (+8) TWF1 Isoform 3 of Twinfilin-1 IPI00815767 TXNDC12 Thioredoxin domain-containing protein 12 precursor IPI00026328 TXNL1 Thioredoxin-like protein 1 IPI00305692 (+1) UBE2I SUMO-conjugating enzyme UBC9 IPI00032957 (+2) UBE2L3 Ubiquitin-conjugating enzyme E2 L3 IPI00021347 UBE2N Ubiquitin-conjugating enzyme E2 N IPI00003949 (+1) UCHL1 Ubiquitin carboxyl-terminal hydrolase isozyme L1 IPI00018352 UCHL3 Ubiquitin carboxyl-terminal hydrolase isozyme L3 IPI00011250 (+1) Uncharacterized protein ENSP00000348237 IPI00453476 (+1) VAPA Vesicle-associated membrane protein-associated protein A IPI00170692 (+1) VEGFA vascular endothelial growth factor A isoform a precursor IPI00012567 (+5) VPS26A Vacuolar protein sorting-associated protein 26A IPI00411426 YWHAB Isoform Short of 14-3-3 protein beta/alpha IPI00759832 YWHAE 14-3-3 protein epsilon IPI00000816 YWHAG 14-3-3 protein gamma IPI00220642 YWHAQ 14-3-3 protein theta IPI00018146 YWHAZ 14-3-3 protein zeta/delta IPI00021263 KRT1 Keratin, type II cytoskeletal 1 IPI00220327 (+1) KRT10 Keratin, type I cytoskeletal 10 IPI00009865 (+1) KRT14 Keratin, type I cytoskeletal 14 IPI00384444 KRT16 Keratin, type I cytoskeletal 16 IPI00217963 KRT17 Keratin, type I cytoskeletal 17 IPI00450768 KRT2 Keratin, type II cytoskeletal 2 epidermal IPI00021304 (+1) KRT27 Keratin, type I cytoskeletal 27 IPI00328103 KRT5 Keratin, type II cytoskeletal 5 IPI00009867 KRT6A Keratin, type II cytoskeletal 6A IPI00300725 KRT73 Isoform 1 of Keratin, type II cytoskeletal 73 IPI00174775 (+2) KRT9 Keratin, type I cytoskeletal 9 IPI00019359 (+1) sp_ALBU_BOVIN IPIsp_ALBU_BOVIN

TABLE 3 Proteins identified by MS in glioma cell line SF-539: Protein: Accession # ACTA2 Actin, aortic smooth muscle IPI00008603 (+16) ACYP1 Acylphosphatase-1 IPI00221117 (+1) ACYP2 Acylphosphatase-2 IPI00216461 (+1) C19orf10 UPF0556 protein C19orf10 precursor IPI00056357 COTL1 Coactosin-like protein IPI00017704 CSTB Cystatin-B IPI00021828 CYCS Cytochrome c IPI00465315 (+1) DBI Isoform a 1 of Acyl-CoA-binding protein IPI00010182 (+2) FKBP1A FK506-binding protein 1A IPI00873810 FLG2 Filaggrin-2 IPI00397801 FN1 Isoform 1 of Fibronectin precursor IPI00022418 (+15) HNRNPH3 Isoform 1 of Heterogeneous nuclear ribonucleoprotein H3 IPI00013877 (+3) ISG15 Interferon-induced 17 kDa protein precursor IPI00375631 LGALS3 Galectin-3 IPI00465431 LYZ Lysozyme C precursor IPI00019038 (+1) MIF Macrophage migration inhibitory factor IPI00293276 MT2A Metallothionein-2 IPI00022498 NEDD8 NEDD8 precursor IPI00020008 (+2) PDIA3 Protein disulfide-isomerase A3 precursor IPI00025252 PFN1 Profilin-1 IPI00216691 RBMX Heterogeneous nuclear ribonucleoprotein G IPI00304692 (+1) RPS27A; UBC; UBB ubiquitin and ribosomal protein S27a precursor IPI00179330 (+21) S100A6 Protein S100-A6 IPI00027463 S100A7 Protein S100-A7 IPI00219806 S100A8 Protein S100-A8 IPI00007047 SH3BGRL SH3 domain-binding glutamic acidrich-like protein IPI00025318 SH3BGRL3 Putative uncharacterized protein IPI00010402 (+2) sp_B2MG_HUMAN IPIsp_B2MG_HUMAN TMSB10 Thymosin beta-10 IPI00220827 TXN Thioredoxin IPI00216298 (+1) TXNDC17 Thioredoxin domain-containing protein 17 IPI00646689 UFM1 Ubiquitin-fold modifier 1 precursor IPI00010207 (+1) KPRP Keratinocyte proline-rich protein IPI00514908 KRT1 Keratin, type II cytoskeletal 1 IPI00220327 (+1) KRT10 Keratin, type I cytoskeletal 10 IPI00009865 KRT14 Keratin, type I cytoskeletal 14 IPI00384444 KRT2 Keratin, type II cytoskeletal 2 epidermal IPI00021304 (+1) KRT5 Keratin, type II cytoskeletal 5 IPI00009867 KRT77 Keratin 77 IPI00376379 KRT9 Keratin, type I cytoskeletal 9 IPI00019359 (+1) sp_TRYP_PIG IPIsp_TRYP_PIG

TABLE 4 Proteins identified by MS from Glioma cell line U251 supernatants: Protein: Accession # A1BG Alpha-1B-glycoprotein precursor IPI00022895 A2M Alpha-2-macroglobulin precursor IPI00478003 C3 Complement C3 precursor (Fragment) IPI00783987 FGG Isoform Gamma-B of Fibrinogen gamma chain precursor IPI00021891 (+3) GLUD1 Glutamate dehydrogenase 1, mitochondrial precursor IPI00016801 (+1) HBA2; HBA1 Hemoglobin subunit alpha IPI00410714 (+1) HBB Hemoglobin subunit beta IPI00654755 (+1) HPX Hemopexin precursor IPI00022488 IGHG1 IGHG1 protein IPI00448925 IGHM IGHM protein IPI00477090 IGHV3OR16-13; IGHA1 IGHA1 protein IPI00166866 (+1) LDHB L-lactate dehydrogenase B chain IPI00219217 LOC100133739 Putative uncharacterized protein DKFZp686C15213 IPI00426051 LTF Growth-inhibiting protein 12 IPI00298860 (+3) MAGI1 Isoform 4 of Membrane-associated guanylate kinase, WW and PDZ IPI00382692 domain-containing protein 1 MPO Isoform H17 of Myeloperoxidase precursor IPI00007244 (+2) SERPINA1 Isoform 1 of Alpha-1-antitrypsin precursor IPI00553177 (+1) SERPINA3 Alpha-1-antichymotrypsin precursor IPI00550991 (+1) TF Serotransferrin precursor IPI00022463 (+2) ALB Isoform 1 of Serum albumin precursor IPI00745872 (+1) KRT1 Keratin, type II cytoskeletal 1 IPI00220327 (+1) KRT10 Keratin, type I cytoskeletal 10 IPI00009865 (+1) KRT14 Keratin, type I cytoskeletal 14 IPI00384444 KRT2 Keratin, type II cytoskeletal 2 epidermal IPI00021304 (+1) KRT5 Keratin, type II cytoskeletal 5 IPI00009867 KRT6C Keratin, type II cytoskeletal 6C IPI00299145 KRT9 Keratin, type I cytoskeletal 9 IPI00019359 (+1) sp_ALBU_BOVIN IPIsp_ALBU_BOVIN sp_TRYP_PIG IPIsp_TRYP_PIG

TABLE 5 Proteins identified by MS of supernatants from colon cell line HCC-2998: Protein: Accession # RPS27A; UBC; UBB ubiquitin and ribosomal protein S27a IPI00179330 precursor S100A6 Protein S100-A6 IPI00027463 S100A7 Protein S100-A7 IPI00219806 S100A8 Protein S100-A8 IPI00007047 S100A9 Protein S100-A9 IPI00027462 SERPINB3 Isoform 1 of Serpin B3 IPI00022204 KRT1 Keratin, type II cytoskeletal 1 IPI00220327 KRT10 Keratin, type I cytoskeletal 10 IPI00009865 KRT2 Keratin, type II cytoskeletal 2 epidermal IPI00021304 KRT9 Keratin, type I cytoskeletal 9 IPI00019359 sp_TRYP_PIG IPIsp_TRYP_PIG 24 kDa 14

TABLE 6 Proteins identified by MS of supernatants from hepatic cell line HepG2: Protein: Accession # B2M Beta-2-microglobulin precursor IPI00004656 C19orf10 Uncharacterized protein C19orf10 precursor IPI00056357 CSTB Cystatin-B IPI00021828 CYCS Cytochrome c IPI00465315 HMGA1 Isoform HMG-I of High mobility group protein HMGI/HMG-Y IPI00179700 LGALS3 Galectin-3 IPI00465431 MIF Macrophage migration inhibitory factor IPI00293276 PFN1 Profilin-1 IPI00216691 PPIA; LOC654188; LOC653214 Peptidyl-prolyl cis-trans isomerase A IPI00419585 RNASE4 Ribonuclease 4 precursor IPI00029699 S100A6 Protein S100-A6 IPI00027463 UBC; RPS27A; UBB ubiquitin and ribosomal protein S27a precursor IPI00179330 KRT1 Keratin, type II cytoskeletal 1 IPI00220327 KRT10 Keratin, type I cytoskeletal 10 IPI00009865 KRT16 Keratin, type I cytoskeletal 16 IPI00217963 KRT2 Keratin, type II cytoskeletal 2 epidermal IPI00021304 KRT9 Keratin, type I cytoskeletal 9 IPI00019359

TABLE 7 Proteins identified by MS of supernatants from ovarian cell line CRL-1978: Proteins Identified by MS analysis of Chemorepellant Fractions of Cell Line CRL-1978 Identified Proteins: Accession # ALB Serum albumin IPI00022434 B2M Beta-2-microglobulin precursor IPI00004656 C19orf10 Uncharacterized protein C19orf10 precursor IPI00056357 CST1 Cystatin-SN precursor IPI00305477 CST3 Cystatin-C precursor IPI00032293 CST4 Cystatin-S precursor IPI00032294 CYCS Cytochrome c IPI00465315 FAM3C Protein FAM3C precursor IPI00021923 ISG15 Interferon-induced 17 kDa protein precursor IPI00375631 KRT1 Keratin, type II cytoskeletal 1 IPI00220327 KRT10 Keratin, type I cytoskeletal 10 IPI00009865 KRT14 Keratin, type I cytoskeletal 14 IPI00384444 KRT2 Keratin, type II cytoskeletal 2 epidermal IPI00021304 KRT9 Keratin, type I cytoskeletal 9 IPI00019359 PFN1 Profilin-1 IPI00216691 PPIA; LOC654188; LOC653214 Peptidyl-prolyl cis-trans IPI00419585 isomerase A PPIB peptidylprolyl isomerase B precursor IPI00646304 S100A6 Protein S100-A6 IPI00027463 TXN Thioredoxin IPI00216298 UBC; RPS27A; UBB ubiquitin and ribosomal protein S27a IPI00179330 precursor

TABLE 8 Proteins identified by MS of supernatants from prostate cell line PC3 and ovarian cell line CRL-1978: Proteins Identified by MS analysis of Chemorepellant Fractions of Cell Line PC3 Identified Proteins: Accession # AGR2 AGR2 IPI00007427 ALB Serum albumin IPI00022434 ARMET ARMET protein precursor IPI00328748 C7orf24 Uncharacterized protein C7orf24 IPI00031564 COTL1 Coactosin-like protein IPI00017704 FAM3C Protein FAM3C precursor IPI00021923 HNRPA2B1 Isoform B1 of Heterogeneous nuclear IPI00396378 ribonucleoproteins A2/B1 HSPG2 Basement membrane-specific heparan sulfate IPI00024284 proteoglycan core protein precursor KRT1 Keratin, type II cytoskeletal 1 IPI00220327 KRT10 Keratin, type I cytoskeletal 10 IPI00009865 KRT14 Keratin, type I cytoskeletal 14 IPI00384444 KRT16 Keratin, type I cytoskeletal 16 IPI00217963 KRT2 Keratin, type II cytoskeletal 2 epidermal IPI00021304 KRT5 Keratin, type II cytoskeletal 5 IPI00009867 KRT6A Keratin, type II cytoskeletal 6A IPI00300725 KRT9 Keratin, type I cytoskeletal 9 IPI00019359 LCN2 Neutrophil gelatinase-associated lipocalin precursor IPI00299547 LMNA Isoform A of Lamin-A/C IPI00021405 NME1; NME1-NME2; NME2 NME1-NME2 protein IPI00795292 NPC2 Epididymal secretory protein E1 precursor IPI00301579 PARK7 Protein DJ-1 IPI00298547 PEBP1 Phosphatidylethanolamine-binding protein 1 IPI00219446 PPIA; LOC654188; LOC653214 Peptidyl-prolyl cis-trans IPI00419585 isomerase A PPIB peptidylprolyl isomerase B precursor IPI00646304 PRDX1 Peroxiredoxin-1 IPI00000874 PRDX6 Peroxiredoxin-6 IPI00220301 RBP4 Plasma retinol-binding protein precursor IPI00022420 TAGLN2 Transgelin-2 IPI00550363 TFF2 Trefoil factor 2 precursor IPI00010675 TIMP2 Metalloproteinase inhibitor 2 precursor IPI00027166 TPT1 Tumor protein, translationally-controlled 1 IPI00009943

TABLE 9 Proteins identified by MS of supernatants from breast cancer cell line SK-BR-3: Protein: Accession # TRFE_HU Serotransferrin precursor (Transferrin) P02787 (Siderophilin) EF1G_HU Elongation factor 1-gamma (EF-1-gamma) P26641 LG3BP_HU galectin 3 binding protein precursor (Lectin Q08380 galactoside-binding soluble 3-binding protein)

As shown in the figures, the following proteins were identified in chemorepulsive fractions of supernatants from cell lines and/or ovarian cystic fluid were shown to induce negative chemotaxis of neutrophils:

actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-1, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein.

Profilin-1 was identified in chemorepulsive supernatant fractions. As shown in the figures, profilin-2 was shown to induce negative chemotaxis.

Example 3 Chemorepellant Proteins Identified in Multiple Chemorepellant Fractions

Table 10 shows chemorepellant proteins that were isolated from chemorepellant fractions of at least two cells or from a cell line and ovarian cystic fluid (as indicated by an “X”) and were shown to induce chemorepulsion of neutrophils in their purified form (as described in Examples 1 and 2). For example, Actin was identified in the chemorepulsive fractions isolated from the supernatant of SF-539 cells and from ovarian cystic fluid sample (described in Example 1).

TABLE 10 Proteins identified in the chemorepellant fractions of at least two cell lines Proteins isolated from Cell Line/Tumor supernatants CRL-1978 PC-3 SF-539 HepG2 786-O ACHN OCI-856 ACTA2 (Actin, aortic X X smooth muscle) B2M (beta-2 X X X microglobulin precursor) CFL1 (Cofilin-1) X X CSTB (cystatin B) X X CYCS (cytochrome C) X X X LGAL3 (galectin-3) X X MIF (macrophage X X migration inhibitory factor) PARK7 Protein DJ-1 X X X PSPH (phosphoserine X X phosphatase) SOD1 (superoxide X X dismutase TXN (thioredoxin) X X YWHAE 14-3-3 epsilon X X YWHAZ (14-3-3 X X X zeta/delta)

Table 11 lists proteins identified in chemorepellant fractions of at least two cell lines or at least one cell line and ovarian cyst fluid.

TABLE 11A Proteins identified in chemorepellant fractions of at least two cell lines or ovarian cystic fluid and at least one cell line OCI-856 CRL- HCC- (Cyst Protein Name 1978 PC-3 SF-539 HepG2 SK-BR-3 2998 786-O ACHN U-251 Fluid) ACBD3 Golgi X X resident protein GCP60 APOA1BP Isoform 1 X X of Apolipoprotein A- I-binding protein precursor ARHGDIA Rho X X GDP-dissociation inhibitor 1 ARMET ARMET X X X protein precursor C19orf10 X X X Uncharacterized protein C19orf10 precursor C19orf33 Isoform 1 X X of Immortalization up-regulated protein C1orf128 Isoform 1 X X of UPF0424 protein C1orf128 C7orf24 X X Uncharacterized protein C7orf24 CALD1 Isoform 4 of X X Caldesmon CALM2; CALM1; CALM3 X X Calmodulin CFL1 Cofilin-1 X X CFL2 Cofilin-2 X X CIAPIN1 Isoform 3 X X of Anamorsin CNPY2 Isoform 1 of X X Protein canopy homolog 2 precursor COTL1 Coactosin- X X like protein CRK v-crk sarcoma X X virus CT10 oncogene homolog isoform b CSTB Cystatin-B X X CYCS Cytochrome c X X X CYR61 CYR61 X X protein DSTN Destrin X X DTD1 D-tyrosyl- X X tRNA(Tyr) deacylase 1 EEF1G Elongation X X X factor 1-gamma EIF4B Eukaryotic X X translation initiation factor 4B EIF6 Eukaryotic X X translation initiation factor 6 FAHD1 Isoform 2 of X X Fumarylacetoacetate hydrolase domain- containing protein 1 FAM3C Protein X X X FAM3C precursor FER1L3 Isoform 1 X X of Myoferlin GLO1 X X Lactoylglutathione X X lyase GSTP1 Glutathione X X S-transferase P HDDC2 Isoform 2 of X X HD domain- containing protein 2 HMGA1 Isoform X X HMG-I of High mobility group protein HMGI/HMG-Y HMGN1 Non- X X histone chromosomal protein HMG-14 HN1 Isoform 1 of X X Hematological and neurological expressed 1 protein HNRNPA2B1 X X Isoform B1 of Heterogeneous nuclear ribonucleoproteins A2/B1 HPRT1 X X Hypoxanthine- guanine phosphoribosyltransferase ISG15 Interferon- X X induced 17 kDa protein precursor KIAA0174 Isoform 1 X X of Uncharacterized protein KIAA0174 LDHB L-lactate X X dehydrogenase B chain LGALS3 Galectin-3 X X LMNA Isoform A of X X Lamin-A/C M6PRBP1 Isoform X X A of Mannose-6- phosphate receptor- binding protein 1 MAPRE1 X X Microtubule- associated protein RP/EB family member 1 NME2 Nucleoside X X diphosphate kinase NPC2 Epididymal X X X secretory protein E1 precursor NQO2 X X Ribosyldihydronicotinamide dehydrogenase NUDT1 Isoform p26 X X of 7,8-dihydro-8- oxoguanine triphosphatase PDAP1 28 kDa X X heat- and acid- stable phosphoprotein PEBP1 X X Phosphatidylethanol amine-binding protein 1 PFN1 Profilin-1 X X X PPIA; LOC654188; LOC653214 X X X X Peptidyl- prolyl cis-trans isomerase A PPIB peptidylprolyl X X X X isomerase B precursor PPIF Peptidyl-prolyl X X cis-trans isomerase, mitochondrial precursor PRDX1 X X X Peroxiredoxin-1 PRDX3 Thioredoxin- X X dependent peroxide reductase, mitochondrial precursor PRDX6 X X Peroxiredoxin-6 QDPR X X Dihydropteridine reductase RAB11B Ras- X X related protein Rab- 11B REXO2 Isoform 1 of X X Oligoribonuclease, mitochondrial precursor (Fragment) RNASET2 Isoform 1 X X of Ribonuclease T2 precursor RPE Isoform 1 of X X Ribulose-phosphate 3-epimerase RPIA Ribose-5- X X phosphate isomerase RPS27A; UBC; UBB X X ubiquitin and ribosomal protein S27a precursor S100A11 Protein X X S100-A11 S100A6 Protein X X X X X S100-A6 S100A7 Protein X X S100-A7 S100A8 Protein X X S100-A8 SCYE1 X X Multisynthetase complex auxiliary component p43 SNX12 Isoform 1 of X X Sorting nexin-12 STX7 Isoform 1 of X X Syntaxin-7 SUB1 Activated X X RNA polymerase II transcriptional coactivator p15 TAGLN2 Transgelin-2 X X TPI1 Isoform 1 of X X Triosephosphate isomerase TPK1 Thiamin X X pyrophosphokinase 1 TPT1 X X Translationally- controlled tumor protein TRFE Human X X X Serotransferrin precursor (Transferrin) (Siderophilin) TWF1 Isoform 3 of X X Twinfilin-1 TXNDC12 X X Thioredoxin domain- containing protein 12 precursor UBC; RPS27A; UBB X X ubiquitin and ribosomal protein S27a precursor UBE2I SUMO- X X conjugating enzyme UBC9 UBE2L3 Ubiquitin- X X conjugating enzyme E2 L3 UCHL1 Ubiquitin X X carboxyl-terminal hydrolase isozyme L1 VAPA Vesicle- X X associated membrane protein- associated protein A YWHAB Isoform X X Short of 14-3-3 protein beta/alpha YWHAE 14-3-3 X X protein epsilon YWHAZ 14-3-3 X X X protein zeta/delta

TABLE 11B Accession numbers for proteins listed in Table 11A Accession # Protein Name IPI00009315 ACBD3 Golgi resident protein GCP60 IPI00168479 (+1) APOA1BP Isoform 1 of Apolipoprotein A-I-binding protein precursor IPI00003815 (+1) ARHGDIA Rho GDP-dissociation inhibitor 1 IPI00328748 ARMET ARMET protein precursor IPI00056357 C19orf10 Uncharacterized protein C19orf10 precursor IPI00030767 C19orf33 Isoform 1 of Immortalization up-regulated protein IPI00015351 C1orf128 Isoform 1 of UPF0424 protein C1orf128 IPI00031564 C7orf24 Uncharacterized protein C7orf24 IPI00218696 CALD1 Isoform 4 of Caldesmon IPI00075248 (+2) CALM2; CALM1; CALM3 Calmodulin IPI00012011 CFL1 Cofilin-1 IPI00413344 CFL2 Cofilin-2 IPI00025333 (+1) CIAPIN1 Isoform 3 of Anamorsin IPI00443909 CNPY2 Isoform 1 of Protein canopy homolog 2 precursor IPI00017704 COTL1 Coactosin-like protein IPI00305469 CRK v-crk sarcoma virus CT10 oncogene homolog isoform b IPI00021828 CSTB Cystatin-B IPI00465315 CYCS Cytochrome c IPI00006273 (+2) CYR61 CYR61 protein IPI00473014 DSTN Destrin IPI00152692 DTD1 D-tyrosyl-tRNA(Tyr) deacylase 1 IPI00000875 (+1) EEF1G Elongation factor 1-gamma IPI00012079 (+1) EIF4B Eukaryotic translation initiation factor 4B IPI00010105 EIF6 Eukaryotic translation initiation factor 6 IPI00440828 (+2) FAHD1 Isoform 2 of Fumarylacetoacetate hydrolase domain- containing protein 1 IPI00021923 FAM3C Protein FAM3C precursor IPI00021048 (+5) FER1L3 Isoform 1 of Myoferlin IPI00220766 GLO1 Lactoylglutathione lyase IPI00219757 (+1) GSTP1 Glutathione S-transferase P IPI00386751 (+1) HDDC2 Isoform 2 of HD domain-containing protein 2 IPI00179700 HMGA1 Isoform HMG-I of High mobility group protein HMGI/HMG-Y IPI00554761 HMGN1 Non-histone chromosomal protein HMG-14 IPI00007764 (+1) HN1 Isoform 1 of Hematological and neurological expressed 1 protein IPI00396378 HNRNPA2B1 Isoform B1 of Heterogeneous nuclear ribonucleoproteins A2/B1 IPI00218493 HPRT1 Hypoxanthine-guanine phosphoribosyltransferase IPI00375631 ISG15 Interferon-induced 17 kDa protein precursor IPI00024660 (+2) KIAA0174 Isoform 1 of Uncharacterized protein KIAA0174 IPI00219217 LDHB L-lactate dehydrogenase B chain IPI00465431 LGALS3 Galectin-3 IPI00021405 (+4) LMNA Isoform A of Lamin-A/C IPI00106668 (+1) M6PRBP1 Isoform A of Mannose-6-phosphate receptor- binding protein 1 IPI00017596 MAPRE1 Microtubule-associated protein RP/EB family member 1 IPI00604590 (+1) NME2 Nucleoside diphosphate kinase IPI00301579 NPC2 Epididymal secretory protein E1 precursor IPI00219129 (+3) NQO2 Ribosyldihydronicotinamide dehydrogenase IPI00004392 (+4) NUDT1 Isoform p26 of 7,8-dihydro-8-oxoguanine triphosphatase IPI00013297 PDAP1 28 kDa heat- and acid-stable phosphoprotein IPI00219446 PEBP1 Phosphatidylethanolamine-binding protein 1 IPI00216691 PFN1 Profilin-1 IPI00419585 PPIA; LOC654188; LOC653214 Peptidyl-prolyl cis-trans isomerase A IPI00646304 PPIB peptidylprolyl isomerase B precursor IPI00026519 PPIF Peptidyl-prolyl cis-trans isomerase, mitochondrial precursor IPI00000874 (+1) PRDX1 Peroxiredoxin-1 IPI00024919 (+1) PRDX3 Thioredoxin-dependent peroxide reductase, mitochondrial precursor IPI00220301 PRDX6 Peroxiredoxin-6 IPI00014439 QDPR Dihydropteridine reductase IPI00020436 (+2) RAB11B Ras-related protein Rab-11B IPI00032830 (+1) REXO2 Isoform 1 of Oligoribonuclease, mitochondrial precursor (Fragment) IPI00414896 (+1) RNASET2 Isoform 1 of Ribonuclease T2 precursor IPI00335280 (+1) RPE Isoform 1 of Ribulose-phosphate 3-epimerase IPI00026513 (+1) RPIA Ribose-5-phosphate isomerase IPI00179330 RPS27A; UBC; UBB ubiquitin and ribosomal protein S27a precursor IPI00013895 S100A11 Protein S100-A11 IPI00027463 S100A6 Protein S100-A6 IPI00219806 S100A7 Protein S100-A7 IPI00007047 S100A8 Protein S100-A8 IPI00006252 (+1) SCYE1 Multisynthetase complex auxiliary component p43 IPI00438170 (+2) SNX12 Isoform 1 of Sorting nexin-12 IPI00289876 (+1) STX7 Isoform 1 of Syntaxin-7 IPI00221222 SUB1 Activated RNA polymerase II transcriptional coactivator p15 IPI00550363 TAGLN2 Transgelin-2 IPI00465028 TPI1 Isoform 1 of Triosephosphate isomerase IPI00072523 (+1) TPK1 Thiamin pyrophosphokinase 1 IPI00550900 TPT1 Translationally-controlled tumor protein IPI00022463 (+2) TRFE Human Serotransferrin precursor (Transferrin) (Siderophilin) IPI00815767 TWF1 Isoform 3 of Twinfilin-1 IPI00026328 TXNDC12 Thioredoxin domain-containing protein 12 precursor IPI00179330 UBC; RPS27A; UBB ubiquitin and ribosomal protein S27a precursor IPI00032957 (+2) UBE2I SUMO-conjugating enzyme UBC9 IPI00021347 UBE2L3 Ubiquitin-conjugating enzyme E2 L3 IPI00018352 UCHL1 Ubiquitin carboxyl-terminal hydrolase isozyme L1 IPI00170692 (+1) VAPA Vesicle-associated membrane protein-associated protein A IPI00759832 YWHAB Isoform Short of 14-3-3 protein beta/alpha IPI00000816 YWHAE 14-3-3 protein epsilon IPI00021263 YWHAZ 14-3-3 protein zeta/delta

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 

1. A method of inducing migration of an immune cell toward a cancer cell comprising inhibiting the activity of a chemorepellant released from the cancer cell. 2-13. (canceled)
 14. The method of claim 1, wherein the chemorepellant has substantial identity to a protein selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein, or a biologically active fragment of any of thereof.
 15. The method of claim 1, wherein the activity of at least two chemorepellant proteins is suppressed.
 16. The method of claim 1, wherein the activity of the chemorepellant is inhibited by the administration of an antibody that binds and inhibits the activity of the chemorepellant, by the administration of an antisense nucleic acid or by the administration of a small molecule.
 17. The method of claim 1, wherein the cancer cell is selected from the group consisting of a colon cancer cell, prostate cancer cell, breast cancer cell, lung cancer cell, skin cancer cell, liver cancer cell, bone cancer cell, pancreas cancer cell, ovarian cancer cell, testicular cancer cell, bladder cancer cell, kidney cancer cell, brain cancer cell, glioma cell, head and neck cancer cell.
 18. The method of claim 17, wherein the cancer is an ovarian cancer cell and chemorepellant protein is selected from the group consisting of 14-3-3 zeta/delta, hemopexin, actin and apolipoprotein A1.
 19. The method of claim 17, wherein the cancer cell is a renal carcinoma cell and the chemorepellant is selected from the group consisting of PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3 gamma, phosphoserine phosphatase and superoxide dismutase.
 20. The method of claim 17, wherein the cancer cell is a glioma cell and the chemorepellant is selected from the group consisting of profilin-2, beta-2 microglobulin, cytochrome C, cystatin B, macrophage migration inhibitory factor, FK506 binding protein, thioredoxin and galectin-3.
 21. A method for the treatment of cancer comprising inducing the migration of an immune cell toward a cancer cell by inhibiting the activity of a chemorepellant released from the cancer cell.
 22. The method of claim 21, wherein the cancer is selected from the group consisting of colon, prostate, breast, lung, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, brain, head and neck cancer.
 23. The method of claim 21, wherein the activity of the chemorepellant is inhibited by the administration of a therapeutically effective amount of an agent that inhibits the activity of the chemorepellant.
 24. The method of claim 23, wherein the agent is an antibody that binds and inhibits the activity of the chemorepellant or is an antisense nucleic acid.
 25. (canceled)
 26. (canceled)
 27. The method of claim 21, wherein the chemorepellant is selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein, or a biologically active fragment thereof.
 28. A method of inducing negative chemotaxis of a human migratory cell comprising administering an effective amount of a chemorepellant, wherein the chemorepellant comprises an amino acid sequence that has substantial identity to a protein isolated from ovarian cancer cystic fluid or from the supernatant of a cancer cell culture wherein the cancer cell is selected from the group consisting of a human renal adenocarcinoma cell line, a human renal carcinoma cell line, human glioblastoma cell line, human colon carcinoma cell line, human hepatocellular carcinoma cell line, human ovary clear carcinoma cell line and human prostate cancer cell line, or to a biologically active fragment of any of thereof, wherein the isolated protein or fragment is capable of inducing chemorepulsion of an immune cell.
 29. (canceled)
 30. The method of claim 28, wherein the chemorepellant is selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein, or a biologically active fragment of any of thereof.
 31. The method of claim 28, wherein the human migratory cell is an immune cell.
 32. The method of claim 31, wherein the immune cell is selected from the group consisting of lymphocytes, monocytes, neutrophils, eosinophils, mast cells, Natural killer cells, dendritic cells, and T cells.
 33. (canceled)
 34. A method of inhibiting the chemotactic induction of an immune cell in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a chemorepellant wherein the chemorepellant comprises an amino acid sequence that has substantial identity to a protein isolated from ovarian cancer cystic fluid or from the supernatant of a cancer cell culture wherein the cancer cell is selected from the group consisting of a human renal adenocarcinoma cell line, a human renal carcinoma cell line, human glioblastoma cell line, human colon carcinoma cell line, human hepatocellular carcinoma cell line, human ovary clear carcinoma cell line and human prostate cancer cell line, or a biologically active fragment of any of thereof, wherein the isolated protein or fragment thereof is capable of inducing chemorepulsion of an immune cell.
 35. The method of claim 34, wherein the chemorepellant is selected from the group consisting of actin, 14-3-3 zeta/delta, apolipoprotein A1, hemopexin, PARK7, cofilin-1, 14-3-3 epsilon, 14-3-3-gamma, phosphoserine phosphatase, superoxide dismutase, profilin-2, beta-2 microglobulin, cytochrome c, cystatin B, macrophage migration inhibitory factor (MIF), FK506 binding protein, thioredoxin, galectin 3, human transferrin, human EF-1-gamma and human galectin 3 binding protein, or a biologically active fragment of any of thereof.
 36. The method of claim 34, wherein the patient is suffering from an inflammatory condition.
 37. (canceled)
 38. The method of claim 34, wherein chemotaxis toward a medical implant is inhibited.
 39. The method of claim 34, wherein chemotaxis toward a transplant or graft is inhibited.
 40. The method of claim 34 wherein the chemorepellant is administered locally. 